Mechanism of catalpol regulating Th17 cell differentiation by interfering PKM2/LDHA expression / 中国药房

OBJECTIVE To investigate the mechanism of catalpol affecting the differentiation of helper T cell 17 （Th17） by interfering the expressions of pyruvate kinase M2 （PKM2） and lactate dehydrogenase A （LDHA）. METHODS The naive CD4+ T cells were selected from the spleen of C57BL/6 mice， and were differentiated into Th17 cells by adding directional differentiation stimulants for 72 hours. At the same time， the cells were treated with 0 （directed control）， 20， 40 and 80 μg/mL catalpol. The flow cytometry was used to detect the proportion of Th17 cell differentiation in cells； the colorimetric method was adopted to detect the levels of pyruvate and lactate in cell culture supernatant； mRNA expressions of retinoid-related orphan nuclear receptor gamma t （RORγt）， PKM2 and LDHA were detected by qRT-PCR method； Western blot was used to detect the expression levels of PKM2， LDHA， signal transducer and activator of transcription 3 （STAT3）， and phosphorylated STAT3 （p-STAT3） proteins in cells. RESULTS Compared with the directed control group， after 72 hours of treatment with 20， 40， 80 μg/mL catalpol， the differentiation ratio of Th17 cells were decreased by 6.74%， 8.41%， 9.24%， and the levels of pyruvate and lactate in the cell culture supernatant， the mRNA expressions of PKM2， LDHA and RORγt as well as the protein expressions of PKM2 and LDHA and the phosphorylation of STAT3 were significantly reduced （P＜0.05）. CONCLUSIONS Catalpol can reduce the glycolysis level by down-regulating the expressions of PKM2 and LDHA， thereby inhibiting the differentiation of Th17 cells.

Knockdown of PTEN promotes colon cancer progression and induces M2 macrophage polarization in the colon cancer cell environment

Objective: This article aims to study the effect of phosphate and tension homolog deleted on chromosome ten (PTEN) knockdown on colon cancer progression and macrophage polarization in the cancer environment. Materials and Methods and Results: The expression of PTEN in colon cancer tissues and colon cancer cells was significantly lower than in precancerous tissues or CCD-18Co cells, and the decrease was most evident in SW620 cells. The expressions of phosphate (p)-p38, c-Jun N-terminal kinase (JNK), activator protein 1 (AP-1), B-cell lymphoma-2 (Bcl-2) protein in colon cancer tissues and cells were significantly higher than in precancerous tissues or CCD-18Co cells (P-values < 0.05). Bcl-2-associated X (Bax) and Caspase-3 expressions in colon cancer tissues and cells were significantly lower than in precancerous tissues or CCD-18Co cells (P-values < 0.05). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was applied to measure cell viability. Transwell evaluated the cell migration and invasion ability. Si-PTEN improved the proliferation, migration, and invasion of SW620 cells (P-values < 0.05). The expression levels of arginase-1 (Arg-1), CD163, CD206 in colon cancer tissues were significantly higher than in precancerous tissues (P-values < 0.05). The cell cycle, the number of M1 and M2 double-positive cells were assessed by flow cytometry. Si-PTEN reduced the expression of tumor necrosis factor-alpha (TNF-?), interleukin-1beta (IL-1?), and inducible nitric oxide synthase (iNOS), which upregulated the expression of Arg-1, CD206, CD163, p-p38, JNK, and AP-1 (P-values < 0.05). Conclusion: Si-PTEN promoted colon cancer progression and induced the polarization of M2 tumor-associated macrophages in the colon cancer cell environment.

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.

Expression of serum connective tissue growth factor, glyoxalase Ⅰ, pyruvate kinase M2 in endometrial carcinoma and its relationship with clinicopathological features / 中国医师进修杂志

Objective:To explore the expression of serum connective tissue growth factor (CTGF), glyoxalase Ⅰ (GLO-I) and pyruvate kinase M2 (PKM2) in endometrial cancer and their relationship with clinicopathological characteristics.Methods:A total of 96 endometrial cancer patients in Yuechi County People's Hospital from February 2015 to February 2017 were selected as the research group, 48 patients with endometrial hyperplasia during the same period were selected as the benign control group, and 48 patients with healthy physical examination during the same period were selected as the healthy control group. The serum levels of CTGF, GLO-Ⅰ, and PKM2 in the three groups were analyzed. The correlation between serum levels of CTGF, GLO-Ⅰ and PKM2 in the research group was analyzed, and the relationship between each serum index and clinicopathological characteristics was analyzed.Results:The levels of serum CTGF, GLO-Ⅰ and PKM2 in the research group were higher than those in the benign control group and healthy control group: (184.31 ± 37.14) μg/L vs. (110.45 ± 20.59), (17.28 ± 0.42) μg/L; (95.17 ± 16.56) pmol/L vs. (56.29 ± 10.14), (9.08 ± 0.66) pmol/L; (20.25 ± 6.13) μg/L vs. (13.11 ± 4.58), (9.05 ± 2.74) μg/L; and the levels of serum CTGF, GLO-Ⅰ and PKM2 in the benign control group were higher than those in the healthy control group, there were statistical differences ( P<0.05). The results of Pearson correlation analysis showed that the level of CTGF had positive correlation with GLO-Ⅰ and PKM2 ( r = 0.713, 0.741, P<0.05), and the level of GLO-Ⅰ had positive correlation with PKM2 ( r = 0.823, P<0.05). The results of Spearman correlation analysis showed that the levels of CTGF, GLO-Ⅰ, PKM2 had positive correlation with FIGO stage ( r = 0.609, 0.704, 0.721; P<0.05), myometrial invasion depth ( r = 0.753, 0.695, 0.719; P<0.05), lymph node metastasis ( r = 0.776, 0.744, 0.640; P<0.05); had negative correlation with the degree of differentiation ( r = - 0.711, - 0.720, - 0.668; P<0.05). Conclusions:Serum CTGF, GLO-I, PKM2 expression levels are abnormally elevated in patients with endometrial cancer, which are significantly related to multiple clinicopathological characteristics.

Recombinant Human Peroxiredoxin-5 Improves Anti-tumor Immunity by Inducing Macrophage Polarization / 中国生物化学与分子生物学报

Tumor cells can use different strategies to suppress the immune system and disable them for killing tumor cells. Previous studies have shown that recombinant human peroxiredoxin-5 (hPRDX5) can activate the normal anti-tumor immune, so as to control and eliminate the tumor cells, but its exact mechanism of action needs to be studied in depth. The study aimed to investigate whether hPRDX5 exerts its anti-tumor activity by activating or reversing the polarization state of mouse macrophages RAW264. 7 cells. The results of CCK8 showed that different doses of hPRDX5 could significantly enhance the viability of macrophage compared with the control group (P < 0. 001); The results of Nitric oxide (NO) test showed that hPRDX5 significantly enhanced NO secretion levels in RAW264. 7 cells (P < 0. 001); ELISA experiments revealed that hPRDX5 promotes TNF-α (P<0. 01) and IL-6 (P<0. 001) secretion in RAW264. 7 cells; Flow cytometry revealed that hPRDX5 increased the expression of antigen differentiation cluster (CD) 80 (P < 0. 01) and inducible nitric oxide oxide synthase (iNOS) (P < 0. 001) in RAW264. 7 cells, and reduced the expression of CD206 (P < 0. 001) in RAW264. 7 cells induced by tumor conditional culture solution (TCS); Lactate dehydrogenase (LDH) experiments revealed that hPRDX5 can increase the killing activity of mouse macrophages on mouse pancreatic cancer Panc02 cells. hPRDX5 is able to activate mouse macrophage RAW264. 7 cells, promotes its M1-type polarization, reverses M2-type polarization, and exerts antitumor activity through the immune-enhancing effect.

Digoxin alleviates pulmonary fibrosis by inhibiting macrophage M2 polarization via regulating mTORC2-IRF4 signaling pathway / 中国药理学通报

Aim To investigate the protective effect of digoxin (Dig) on the bleomycin (BLM)-induced pulmonary fibrosis in mice and the underlying mechanism. Methods Pulmonary fibrosis model was established by intratracheal instillation of BLM (5 mg · kg

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.

Mechanism of M2 macrophage-derived exosomes in promoting the migration of glioblastoma via transferring miR-1260b / 中国药科大学学报

@#Tumor-associated macrophage promotes the progression of glioblastoma (GBM) by infiltrating into tumor tissue, yet its mechanism has not been fully elucidated.This paper aimed to investigate the mechanism of M2 macrophages in affecting the migratory capacity of GBM via secreting exosomes.Ultracentrifugation was used to extract exosomes; RNA sequencing was carried out to screen differentially expressed miRNAs; target prediction database was used to predict the possible target proteins of miRNA; Dual-luciferase reporter assay was performed to verify the interaction between miRNA and target genes; and the proliferation ability of tumor cells was detected by subcutaneous xenograft model in nude mice.Results showed that tumor-related macrophages were mainly M2 macrophages, and that exosomes secreted by M2 macrophages could promote the migration of glioma cells.Meanwhile, exosomes secreted by M2 macrophages transported miR-1260b and affected the migration of glioma cells through directly targeted AJAP1, suggesting that exosomes secreted by macrophages could affect the migration ability of GBM through transporting miR-1260b.

Phosphorylated PKM2 regulates endothelium-dependent vasodilation in diabetes / 中南大学学报(医学版)

OBJECTIVES@#Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy. The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells. Glycolysis pathway is the major energy source for endothelial cells. Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose. Pyruvate kinase isozyme type M2 (PKM2) is one of key enzymes in glycolysis pathway, phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose. TEPP-46 can stabilize PKM2 in its tetramer form, reducing its dimer formation and phosphorylation. Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation, this study aims to explore the impact and potential mechanism of phosphorylated PKM2 (p-PKM2) on endothelial dependent vasodilation function in high glucose, and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy.@*METHODS@#The mice were divided into 3 groups: a wild-type (WT) group (a control group, C57BL/6 mice) and a db/db group (a diabetic group, db/db mice), which were treated with the sodium carboxymethyl cellulose solution (solvent) by gavage once a day, and a TEPP-46 group (a treatment group, db/db mice+TEPP-46), which was gavaged with TEPP-46 (30 mg/kg) and sodium carboxymethyl cellulose solution once a day. After 12 weeks of treatment, the levels of p-PKM2 and PKM2 protein in thoracic aortas, plasma nitric oxide (NO) level and endothelium-dependent vasodilation function of thoracic aortas were detected. High glucose (30 mmol/L) with or without TEPP-46 (10 μmol/L), mannitol incubating human umbilical vein endothelial cells (HUVECs) for 72 hours, respectively. The level of NO in supernatant, the content of NO in cells, and the levels of p-PKM2 and PKM2 protein were detected. Finally, the effect of TEPP-46 on endothelial nitric oxide synthase (eNOS) phosphorylation was detected at the cellular and animal levels.@*RESULTS@#Compared with the control group, the levels of p-PKM2 in thoracic aortas of the diabetic group increased (P<0.05). The responsiveness of thoracic aortas in the diabetic group to acetylcholine (ACh) was 47% lower than that in the control group (P<0.05), and that in TEPP-46 treatment group was 28% higher than that in the diabetic group (P<0.05), while there was no statistically significant difference in the responsiveness of thoracic aortas to sodium nitroprusside (SNP). Compared with the control group, the plasma NO level of mice decreased in the diabetic group, while compared with the diabetic group, the phosphorylation of PKM2 in thoracic aortas decreased and the plasma NO level increased in the TEPP-46 group (both P<0.05). High glucose instead of mannitol induced the increase of PKM2 phosphorylation in HUVECs and reduced the level of NO in supernatant (both P<0.05). HUVECs incubated with TEPP-46 and high glucose reversed the reduction of NO production and secretion induced by high glucose while inhibiting PKM2 phosphorylation (both P<0.05). At the cellular and animal levels, TEPP-46 reversed the decrease of eNOS (ser1177) phosphorylation induced by high glucose (both P<0.05).@*CONCLUSIONS@#p-PKM2 may be involved in the process of endothelium-dependent vasodilation dysfunction in Type 2 diabetes by inhibiting p-eNOS (ser1177)/NO pathway.

Shikonin induces hepatocellular carcinoma cell apoptosis by suppressing PKM2/PHD3/HIF-1α signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism of shikonin-induced death of human hepatocellular carcinoma SMMC-7721 cells.@*METHODS@#Cultured SMMC-7721 cells and normal hepatocytes (L-02 cells) were treated with 4, 8, or 16 μmol/L shikonin, and the changes in cell viability was assessed using MTT assay. The levels of ATP and lactic acid in the cell cultures were detected using commercial kits. Co-immunoprecipitation and immunofluorescence staining were used to determine the relationship among pyruvate kinase M2 (PKM2), prolyl hydroxylase 3 (PHD3), and hypoxia-inducible factor-1α (HIF-1α). The expressions of PHD3, PKM2, HIF-1α, Bax, cleaved caspase-3, and Bcl-2 in SMMC-7721 cells were detected with Western blotting, and cell apoptosis was analyzed with annexin V-FITC/PI staining. The effects of RNA interference of PKM2 on PHD3 and HIF-1α expressions in SMMC-7721 cells were detected using Western blotting.@*RESULTS@#The IC50 of shikonin against SMMC-7721 and L-02 cells was 8.041 μmol/L and 31.75 μmol/L, respectively. Treatment with shikonin significantly inhibited the protein expressions of PKM2, HIF-1α and PHD3 and nuclear translocation of PKM2 and HIF-1α in SMMC-7721 cells. Coimmunoprecipitation and immunofluorescence staining confirmed that shikonin inhibited the formation of PKM2/PHD3/HIF-1α complex and significantly reduced the contents of lactic acid and ATP in SMMC-7721 cells (P < 0.05). The expressions of PHD3 and HIF-1α decreased significantly after PKM2 knockdown (P < 0.05). Shikonin treatment significantly increased the apoptosis rate, enhanced the expressions of Bax and cleaved caspase-3, and decreased Bcl-2 expression in SMMC-7721 cells (P < 0.05).@*CONCLUSIONS@#Shikonin induces apoptosis of SMMC-7721 cells possibly by inhibiting aerobic glycolysis through the PKM2/PHD3/HIF-1α signaling pathway to cause energy supply dysfunction in the cells.

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.

Effect of PKM2 on Osteogenic and Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Myeloma Bone Disease / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of pyruvate kinase M2 (PKM2) in bone marrow mesenchymal stem cells (BMSCs) in myeloma bone disease (MBD) and its effect on osteogenic and adipogenic differentiation of BMSCs.@*METHODS@#BMSCs were isolated from bone marrow of five patients with multiple myeloma (MM) (MM group) and five with iron deficiency anemia (control group) for culture and identification. The expression of PKM2 protein were compared between the two groups. The differences between osteogenic and adipogenic differentiation of BMSCs were assessed by using alkaline phosphatase (ALP) and oil red O staining, and detecting marker genes of osteogenesis and adipogenesis. The effect of MM cell line (RPMI-8226) and BMSCs co-culture on the expression of PKM2 was explored. Functional analysis was performed to investigate the correlations of PKM2 expression of MM-derived BMSCs with osteogenic and adipogenic differentiation by employing PKM2 activator and inhibitor. The role of orlistat was explored in regulating PKM2 expression, osteogenic and adipogenic differentiation of MM-derived BMSCs.@*RESULTS@#Compared with control, MM-originated BMSCs possessed the ability of increased adipogenic and decreased osteogenic differentiation, and higher level of PKM2 protein. Co-culture of MM cells with BMSCs markedly up-regulated the expression of PKM2 of BMSCs. Up-regulation of PKM2 expression could promote adipogenic differentiation and inhibit osteogenic differentiation of MM-derived BMSCs, while down-regulation of PKM2 showed opposite effect. Orlistat significantly promoted osteogenic differentiation in MM-derived BMSCs via inhibiting the expression of PKM2.@*CONCLUSION@#The overexpression of PKM2 can induce the inhibition of osteogenic differentiation of BMSCs in MBD. Orlistat can promote the osteogenic differentiation of BMSCs via inhibiting the expression of PKM2, indicating a potential novel agent of anti-MBD therapy.

Prognostic Significance of LPCAT1 in Adult Acute Myeloid Leukemia Patients with FAB Subtype M2 / 中国实验血液学杂志

OBJECTIVE@#To study the prognostic value of LPCAT1 in acute myeloid leukemia (AML).@*METHODS@#TaqMan-based reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect relative expression of LPCAT1 in 214 newly diagnosed adult AML patients and 24 normal controls. Survival functions were estimated using the Kaplan-Meier method and were compared by the Log-rank test. A Cox proportional hazard regression model was used to identify prognostic factors.@*RESULTS@#The expression level of LPCAT1 in adult AML was 34.37%(1.83%-392.63%), which was significantly lower than 92.81%(2.60%-325.84%) of normal controls (P<0.001). The prognostic significance of LPCAT1 was evaluated in 171 non-acute promyelocytic leukemia patients with complete clinical information and prognostic data. Survival analysis showed that the expression level of LPCAT1 had no significant effect on the prognosis of the whole cohort. However, in AML patients with FAB subtype M2 (AML-M2), the 2-year relapse-free survival (RFS) rate of patients with low LPCAT1 expression was 35.4%(95%CI: 0.107-0.601), which was significantly lower than 79.2%(95%CI: 0.627-0.957) of patients with high LPCAT1 expression (P=0.012). Multivariate analysis showed that low expression of LPCAT1 was an independent risk factor for RFS of AML-M2 patients (HR=0.355, 95%CI: 0.126-0.966, P=0.049).@*CONCLUSION@#In adult AML patients LPCAT1 shows low expression. Low LPCAT1 expression is an independent risk factor for RFS in M2-AML patients.

Association of Anti-β 1 and Anti-M2 Antibodies with Autonomic Nervous System Modulation in Patients with Chronic Chagas Cardiomyopathy

Abstract Background: Different immune mechanisms of myocardial damage involved in the pathophysiology of Chagas disease coexist with high titers of autoantibodies induced by T. cruzi . There are few studies in the literature about the adaptive role of anti-β1 and anti-M2 antibodies in chronic Chagas cardiomyopathy (CCC). Objectives: To evaluate the association between anti-β1 and anti-M2 antibodies with heart rate variability (HRV) parameters on 24h Holter monitoring and the rate-pressure product (RPP) on cardiopulmonary exercise testing (CPET). Methods: Anti-β1 and anti-M2 antibody titers were measured by enzyme-linked immunosorbent assay (ELISA) in 64 patients affected by CCC. Analysis of HRV was performed through the time-domain indices NNs, mean NN, SDNN, SDANN, SDNN index, NNNs, RMSSD, and pNN50. Spearman's correlation coefficient was used to assess the association between antibody titers and numerical variables. The Mann-Whitney test was used for comparison between two groups. Multiple linear regression was used to identify independent variables capable of explaining anti-β1 and anti-M2 antibody titers at the 5% significance level. Results: On 24h Holter, during the period of greatest parasympathetic activation (2:00-6:00 a.m.), an inverse association was found between anti-β1 titers and SDNN (rs=-0.13, p =0.041, n=43), as well as a direct association between anti-M2 titers and SDANN ( r s=0.317, p =0.039, n=43). Regarding CPET variables, anti-β1 titers were directly associated with RPP (rs=0.371, p =0.005, n=56). The subgroup of patients with a normal chronotropic response showed higher anti-β1 titers than the subgroup with an impaired response (p=0.023). RPP was an independent explanatory variable for anti-β1 titers, although with a low coefficient of determination (R2=0.147). Conclusion: The findings of this study suggest that, in patients with CCC, anti-β1 and anti-M2 antibodies may affect HRV parameters. RPP was directly associated with higher anti-β1 titers.

Prevention of Premature Ovulation by Administration of Gonadotropin Releasing Hormone Antagonist the day After Ovulation Triggering in Diminished Ovarian Reserve Patients / Prevenção da ovulação prematura por meio da administração de antagonismo de liberação de gonadotropismo no dia seguinte à ovulação

Abstract Objective The aim of the present retrospective study was to investigate the effectiveness of single-dose gonadotropin releasing hormone (GnRH) antagonist administration, the day after human chorionic gonadotropin (hCG) triggering for final oocyte maturation, on the prevention of premature luteinization in patients with diminished ovarian reserve in in-vitro fertilization (IVF) cycles. The secondary objective of the study was to search the effect of this protocol on pregnancy outcomes. Methods This is a retrospective study including 267 infertile patients who have single antral follicle seen with ultrasonography on the 2nd or 3rd day of the menstrual cycle before starting IVF treatment. We randomized patients into two groups. The case group comprised patients who had single-dose GnRH antagonist injection the day after hCG triggering formed, and the patients who had the standard treatment regime formed the control group. In both groups, the oocytes were collected 36 hours after hCG injection. Results The premature ovulation rate was significantly low in the case group compared with the control group (6.86 versus 20.6% per scheduled cycle) (p=0.022). Also, the oocyte retrieval rate (93.14 versus 67.87% per scheduled cycle) (p=0.013), the oocyte maturity rate (79.42 versus 47.87%) (p=0.041), the fertilization rate (65.68 versus 34.54%) (p=0.018), and the embryo transfer rate per scheduled cycle (44.11 versus 18.78%) (p=0.003) were higher in the GnRH antagonist group than in the control group. Conclusion The administration of GnRH antagonist the day after hCG trigger in IVF treatments of patients with diminished ovarian reserve enabled a significant decrease in the rate of premature ovulation but had no effect on live birth rate.

Resumo Objetivo O objetivo do presente estudo retrospectivo foi investigar a eficácia da administração do antagonista do hormônio liberador da gonadotrofina (GnRH) em dose única no dia seguinte ao desencadeamento da gonadotrofina coriônica humana (hCG) para a maturação final do oócito, na prevenção da luteinização prematura em pacientes com diminuição do ovário reserva em ciclos de fertilização in vitro (FIV). O objetivo secundário do estudo foi pesquisar o efeito deste protocolo nos resultados da gravidez. Métodos Trata-se de um estudo retrospectivo incluindo 267 pacientes inférteis que apresentam um único folículo antral visto por ultrassonografia no 2° ou 3° dia do ciclo menstrual antes de iniciar o tratamento de FIV. Nós randomizamos os pacientes em dois grupos. Os pacientes que receberam injeção de antagonista de GnRH em dose única no dia seguinte ao desencadeamento do hCG formaram o grupo caso, e os pacientes que receberam o regime de tratamento padrão formaram o grupo controle. Em ambos os grupos, os oócitos foram coletados 36 horas após a injeção de hCG. Resultados A taxa de ovulação prematura foi significativamente baixa no grupo caso em comparação com o grupo controle (6,86 versus 20,6% por ciclo programado) (p=0,022). Além disso, a taxa de recuperação de oócitos (93,14 versus 67,87% por ciclo programado) (p=0,013), a taxa de maturidade do oócito (79,42 versus 47,87%) (p=0,041), a taxa de fertilização (65,68 versus 34,54%) (p=0,018) e a taxa de transferência de embriões por ciclo programado (44,11 versus 18,78%) (p=0,003) foram maiores no grupo antagonista de GnRH do que no grupo controle. Conclusão A administração de antagonista de GnRH, no dia seguinte ao desencadeamento de hCG em tratamentos de FIV de pacientes com reserva ovariana diminuída permitiu uma redução significativa na taxa de ovulação precoce,mas não teve efeito na taxa de nascidos vivos.

Mechanism of sinomenine in regulating M2 macrophage polarization induced by gastric cancer cells / 西安交通大学学报(医学版)

【Objective】 To study the role and mechanism of sinomenine in the macrophage polarization induced by gastric cancer cells. 【Methods】 Sinomenine was added to gastric cancer cells BGC-823 and MKN-45， cell viability was measured by CCK-8， cell proliferation was measured by colony formation experiment， Co-culture and Transwell cell migration experiments were used to evaluate the recruitment and polarization of macrophages by sinomenine， flow cytometry was used to evaluate the polarization of macrophages， and qRT-PCR and Western blot were used to detect the expression of gene RNA and protein levels. 【Results】 Sinomenine could inhibit the proliferation of gastric cancer cells and the recruitment of gastric cancer cells to macrophages， thus promoting macrophage M2 polarization. It simultaneously inhibited the expression of STAT6 as well as the expression and phosphorylation of C/EBPβ. When STAT6 is overexpressed， it could reduce these inhibitory effects of sinomenine on gastric cancer cells. Further research found that STAT6 mediated the secretion of IL-6 by gastric cancer cells， which was the cause of sinomenine-mediated macrophage recruitment and M2 polarization. 【Conclusion】 The natural drug sinomenine has a good tumor-suppressing ability against gastric cancer， directly inhibits the survival and migration of gastric cancer cells， and inhibits the expression of IL-6 and the M2 phenotype in the tumor microenvironment， reshapes the tumor environment， and reduces the risk of M2 type macrophages for gastric cancer tumors.

Basic and clinical treatments target tumor-associated macrophages in liver cancer / 西安交通大学学报(医学版)

Hepatocellular carcinoma （HCC） most often develops in patients with liver disease characterized by chronic non-resolving inflammation. The inflammatory response is mainly derived from innate immune cells， and tumor-associated macrophages （TAMs） play an important role in the development of tumors．It is usually chemotactic from mononuclear progenitor cells in the blood to tumor tissue， then is induced by the tumor microenvironment and further develops into TAMs. They play an important role in promoting tumor growth，angiogenesis and tumor invasion and inducing tumor tissues to form an environment without inhibition mechanisms. As the relationship between TAMs and malignant tumors becomes clearer， TAMs are beginning to be seen as therapeutic targets. The heterogeneity of TAM subtypes and their origin and dynamic phenotype during the initiation and progression of HCC has been partially unraveled. It further forms the base for developing therapeutic agents by decreasing the population of TAMs via blocking recruitment of bone marrow-derived monocytes and functionally reprograms TAMs to anti-tumoral behavior. This review focuses on the preclinical evolution and hitherto clinical trials for TAM-targeted therapy in HCC.

Tetrahydropalmatine alleviated diabetic neuropathic pain by inhibiting activation of microglia via p38 MAPK signaling pathway / 中国中药杂志

Neuropathic pain is one of the common complications of diabetes. Tetrahydropalmatine(THP) is a main active component of Corydalis Rhizoma with excellent anti-inflammatory and pain-alleviating properties. This study aims to investigate the therapeutic effect of THP on diabetic neuropathic pain(DNP) and the underlying mechanism. High-fat and high-sugar diet(4 weeks) and streptozotocin(STZ, 35 mg·kg~(-1), single intraperitoneal injection) were employed to induce type-2 DNP in rats. Moreover, lipopolysaccharide(LPS) was used to induce the activation of BV2 microglia in vitro to establish an inflammatory cellular model. Fasting blood glucose(FBG) was measured by a blood glucose meter. Mechanical withdrawal threshold(MWT) was assessed with von Frey filaments, and thermal withdrawal latency(TWL) with hot plate apparatus. The protein expression levels of OX42, inducible nitric oxide synthase(iNOS), CD206, p38, and p-p38 were determined by Western blot, the fluorescence expression levels of OX42 and p-p38 in the dorsal horn of the rat spinal cord by immunofluorescence, the mRNA content of p38 and OX42 in rat spinal cord tissue by qRT-PCR, and levels of nitric oxide(NO), interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), interleukin-10(IL-10), and serum fasting insulin(FINS) by enzyme-linked immunosorbent assay(ELISA). RESULTS:: showed that the mo-del group demonstrated significant decrease in MWT and TWL, with pain symptoms. THP significantly improved the MWT and TWL of DNP rats, inhibited the activation of microglia and p38 MAPK signaling pathway in rat spinal cord, and ameliorated its inflammatory response. Meanwhile, THP promoted the change of LPS-induced BV2 microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, suppressed the activation of the p38 MAPK signaling pathway, decreased the expression levels of inflammatory factors NO, IL-1β, IL-6, and TNF-α, and increased the expression level of anti-inflammatory factor IL-10. The findings suggested that THP can significantly ameliorate the pain symptoms of DNP rats possibly by inhibiting the inflammatory response caused by M1 polarization of microglia via the p38 MAPK pathway.

Therapeutic effects of hypoxia-inducible factor-1α siRNA on collagen-induced arthritis in mice and its inhibitory effects on M1 macrophages / 中华微生物学和免疫学杂志

Objective:To investigate the therapeutic effects of a small interfering RNA (siRNA) targeting hypoxia inducible factor-1α (HIF-1α) on collagen-induced arthritis (CIA) in mice and to analyze the possible mechanism at the macrophage level.Methods:DBA/1 mice were injected with bovine type Ⅱ collagen to establish the CIA model. Then, they were injected with HIF-1α-siRNA adenovirus or negative control adenovirus through tail vein once a week for four weeks. This study included four groups: control group, CIA model group, negative control group and HIF-1α-siRNA group. Mouse bone marrow-derived macrophages (BMDMs) were isolated and cultured. The relative expression of CD206 and arginine (Arg) at mRNA level in mouse BMDMs was detected by RT-PCR. The proportions of F4/80 + CD16/32 + M1 and F4/80 + CD206 + M2 macrophages in spleen and thymus were detected by flow cytometry. Pathological changes in the ankle joint of mice were observed using HE staining. Immunohistochemistry was used to detect the expression of macrophages and the subsets in mouse synovial tissues. Results:(1) Compared with the control group, the CIA model group showed decreased expression of CD206 at mRNA level in BMDMs, but increased expression of Arg at mRNA level ( P<0.01). HIF-1α-siRNA increased the expression of CD206 at mRNA level ( P<0.05) and reduced the expression of Arg at mRNA level in BMDMs of mice with CIA ( P<0.01). (2) Compared with the control group, the mice in the CIA model group had increased proportions of F4/80 + CD16/32 + M1 macrophages in splenocytes and thymocytes ( P<0.05), but decreased proportions of F4/80 + CD206 + M2 macrophages in thymocytes ( P<0.05). HIF-1α-siRNA could down-regulate the proportions of F4/80 + CD16/32 + M1 macrophages in splenocytes and thymocytes ( P<0.05), and up-regulate the proportions of F4/80 + CD206 + M2 macrophages in thymocytes of CIA mice ( P<0.01). (3) CIA mice had synovial hyperplasia and macrophages infiltration, especially M1 macrophages, in the ankle joint. HIF-1α-siRNA could alleviate the synovial hyperplasia and macrophage infiltration. Conclusions:HIF-1α-siRNA could alleviate macrophage infiltration and synovial hyperplasia in CIA mice through reducing the proportions of M1 macrophages in thymocytes, BMDMs and synovial tissues and increasing the proportion of M2 macrophages, suggesting that HIF-1α-siRNA could treat CIA mice by regulating the differentiation of M1 and M2 macrophages.

Interleukin-27 Disrupts the Crosstalk of Apoptotic Activities between 4T1 Breast Cancer Cells and M2 Macrophages

@#Introduction: Cytokine immunotherapy such as Interleukin-27 (IL-27) has been foreseen as a promising alternative anti-cancer treatment. Thus, this study aimed to investigate whether IL-27 gene therapy regulates crosstalk between breast cancer cells and macrophages in the sense of pro-apoptotic activities. Methods: This study has led to the development of recombinant pcDNA3.4-IL27. The recombinant pcDNA3.4-IL27 was transfected into 4T1 murine mammary carcinoma cells alone and co-culture of 4T1 with M2 macrophages. The successful expression of IL-27 in the cells were determine through the immunofluorescence staining and detection of CD206, M2 macrophages marker. Apoptotic effects of pcDNA3.4-IL27 were assessed through MTT assay, Annexin V flow cytometer analysis, and AO/PI dual staining. Results: Our findings shows that pcDNA3.4-IL27 has the ability to induce apoptosis in both of the cell group and performs better in the co-culture of 4T1 with M2 macrophages compared to 4T1 cells alone. PcDNA3.4-IL27 induced apoptosis through the altered cell morphology and reduction in the number of viable cells. Conclusion: These data demonstrate that pcDNA3.4-IL27 has the ability to induce apoptosis in both 4T1 cell alone and co-cultured 4T1 with M2 macrophages. Thus, could serve as a potential anti cancer candidate against breast cancer.