Knockdown of interferon-γ inducible protein 30 (IFI30) inhibits the proliferation, invasion and migration of human glioma U251 cells by activating STAT1 and promotes their apoptosis / 细胞与分子免疫学杂志

Objective To establish U251 cells with inhibited expression of interferon-γ inducible protein 30 (IFI30), and to investigate the effect of IFI30 on cell biological function as well as its underlying mechanism. Methods Three knockdown sequences which target IFI30 were designed online and 3 small interfering RNAs (siRNA) were synthesized. After transfection, the inhibition efficiency was detected by real-time quantitative PCR. The siRNA sequence with the highest inhibition efficiency was selected to create short hairpin RNA (shRNA) plasmids. The recombinant plasmids and packaging plasmids were co-transfected into HEK293T cells to prepare lentivirus. The glioma U251 cells were transfected with lentivirus, and the positive cells were screened by puromycin. CCK-8 assay, 5-ethyl-2'-deoxyuridine (EdU) and colony formation assays were used to analyze cell proliferation; the flow cytometry was used to analyze cell cycle and apoptosis; the TranswellTM assay was used to detect cell invasion; the wound-healing assay was employed to detect cell migration, and western blot analysis to detect the protein expresison of cyclin D1, B-cell lymphoma factor 2 (Bcl2), epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), signal transducer and activator of transcription 1 (STAT1). Results The sequence which effectively target IFI30 was screened and U251 cell line capable of inhibiting the IFI30 expression was successfully established. When IFI30 expression was knocked down, the proliferation of U251 cells was inhibited, along with increased ratio of cells in the phase G0/G1, the decreased phase S, the increased rate of cell apoptosis. The cell invasion and migration capabilities was also reduced. The decreased expression of cyclin D1, Bcl2 and N-cadherin were observed in U251 cells, and the expression of E-cadherin and the phosphorylation of STAT1 were found increased. Conclusion Knockdown of IFI30 inhibits the proliferation, invasion and migration of human glioma cell U251 and promotes its apoptosis by activating STAT1.

Interferon-α mediating the functional damage of CD56dimCD57+natural killer cells in peripheral blood of systemic lupus erythematosuss / 北京大学学报(医学版)

OBJECTIVE@#To investigate the regulatory effect of interferon-α (IFN-α) on the apoptosis and killing function of CD56dimCD57+ natural killer (NK) cells in systemic lupus erythematosus (SLE) patients, and to explore the specific mechanism.@*METHODS@#A total of sixty-four newly treated SLE patients and sixteen healthy controls (HC) enrolled in the Second Hospital of Dalian Medical University were selected as the research subjects. And the gene expression levels of molecules related to NK cell-killing function were detected by real-time quantitative polymerase chain reaction. CD56dimCD57+ NK cells were co-cultured with the K562 cells, and the apoptotic K562 cells were labeled with Annexin-Ⅴ and 7-amino-actinomycin D. Peripheral blood mononuclear cells were treated with 20, 40, and 80 μmol/L hydrogen peroxide (H2O2), and treated without H2O2 as control, the expression level of perforin (PRF) was detected by flow cytometry. The concentration of IFN-α in serum was determined by enzyme linked immunosorbent assay. The expression levels of IFN-α receptors (IFNAR) on the surface of CD56dimCD57+ NK cells were detected by flow cytometry, and were represented by mean fluorescence intensity (MFI). CD56dimCD57+ NK cells were treated with 1 000 U/mL IFN-α for 24, 48 and 72 h, and no IFN-α treatment was used as the control, the apoptosis and the expression levels of mitochondrial reactive oxygen species (mtROS) were measured by flow cytometry and represented by MFI.@*RESULTS@#Compared with HC(n=3), the expression levels of PRF1 gene in peripheral blood NK cells of the SLE patients (n=3) were decreased (1.24±0.41 vs. 0.57±0.12, P=0.05). Compared with HC(n=5), the ability of peripheral blood CD56dimCD57+ NK cells in the SLE patients (n=5) to kill K562 cells was significantly decreased (58.61%±10.60% vs. 36.74%±6.27%, P < 0.01). Compared with the control (n=5, 97.51%±1.67%), different concentrations of H2O2 treatment significantly down-regulated the PRF expression levels of CD56dimCD57+ NK cells in a dose-dependent manner, the 20 μmol/L H2O2 PRF was 83.23%±8.48% (n=5, P < 0.05), the 40 μmol/L H2O2 PRF was 79.53%±8.56% (n=5, P < 0.01), the 80 μmol/L H2O2 PRF was 76.67%±7.16% (n=5, P < 0.01). Compared to HC (n=16), the serum IFN-α levels were significantly increased in the SLE patients (n=45) with moderate to high systemic lupus erythematosus disease activity index (SLEDAI≥10) [(55.07±50.36) ng/L vs. (328.2±276.3) ng/L, P < 0.001]. Meanwhile, compared with HC (n=6), IFNAR1 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=6) were increased (MFI: 292.7±91.9 vs. 483.2±160.3, P < 0.05), and compared with HC (n=6), IFNAR2 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=7) were increased (MFI: 643.5±113.7 vs. 919.0±246.9, P < 0.05). Compared with control (n=6), the stimulation of IFN-α (n=6) significantly promoted the apoptosis of CD56dimCD57+ NK cells (20.48%±7.01% vs. 37.82%±5.84%, P < 0.05). In addition, compared with the control (n=4, MFI: 1 049±174.5), stimulation of CD56dimCD57+ NK cells with IFN-α at different times significantly promoted the production of mtROS in a time-dependent manner, 48 h MFI was 3 437±1 472 (n=4, P < 0.05), 72 h MFI was 6 495±1 089 (n=4, P < 0.000 1), but there was no significant difference at 24 h of stimulation.@*CONCLUSION@#High serum IFN-α level in SLE patients may induce apoptosis by promoting mtROS production and inhibit perforin expression, which can down-regulate CD56dimCD57+ NK killing function.

The expression of autophagy-related proteins in peripheral blood mononuclear cells of patients with lupus nephritis is upregulated and related to kidney damage / 细胞与分子免疫学杂志

Objective To identify the relationship between nephritis activity, autophagy and inflammation in patients with SLE. Methods Western blot analysis was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3) and P62 in peripheral blood mononuclear cells (PBMCs) of SLE patients with lupus nephritis and non-lupus nephritis patients. Tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) in the serum of SLE patients were determined by ELISA. The correlation between LC3II/LC3I ratio and SLE disease activity score (SLEDAI), urinary protein, TNF-α and IFN-γ levels was analyzed by Pearson method. Results The expression of LC3 was increased and P62 was decreased in SLE patients. TNF-α and IFN-γ were increased in the serum of SLE patients. LC3II/LC3I ratio was positively correlated with SLEDAI (r=0.4560), 24 hour urine protein (r=0.3753), IFN-γ (r=0.5685), but had no correlation with TNF-α (r=0.04 683). Conclusion Autophagy is found in PBMCs of SLE, and the autophagy is correlated with renal damage and inflammation in patients with lupus nephritis.

Recombinant porcine interferon-gamma expressed in CHO cells and its antiviral activity / 生物工程学报

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×107 U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.

Effect of moxibustion at "Mingmen" (GV 4) and "Guanyuan" (CV 4) on immune function in healthy rats based on intestinal flora / 中国针灸

OBJECTIVE@#To observe the effects of moxibustion at "Mingmen" (GV 4) and "Guanyuan" (CV 4) on immune function and intestinal flora in healthy rats, thereby investigating the underlying mechanism of moxibustion on immune function.@*METHODS@#Twenty 8-week-old SD rats were randomly divided into a young blank group and a young moxibustion group, with 10 rats in each group. Similarly, twenty 8-month-old SD rats were randomly divided into a middle-aged blank group and a middle-aged moxibustion group, with 10 rats in each group. The rats in the two moxibustion groups received moxibustion at "Mingmen" (GV 4) and "Guanyuan" (CV 4), 15 min per session, once daily, five times a week, for a total of four months. The rats in the two blank groups were fed under normal conditions. After the intervention, thymus and spleen indexes were calculated; the morphology of thymus and spleen tissues was observed using HE staining; the flow cytometry was used to detect the expression of CD and CD T lymphocytes and the CD/CD ratio was calculated; ELISA was used to measure the serum levels of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-17 (IL-17); 16S rRNA high-throughput sequencing was used to analyze the intestinal flora. Additionally, the correlation between the relative abundance of intestinal flora and serum levels of TNF-α, IFN-γ, IL-6, IL-10 and IL-17 was analyzed.@*RESULTS@#Compared with the young blank group, the young moxibustion group exhibited an increase in the cortical area of thymus tissue with tighter lymphocyte arrangement; compared with the middle-aged blank group, the middle-aged moxibustion group showed an increase in thymus index (P<0.05) and an increase in the cortical area of thymus tissue. There were no significant differences in spleen index between the 2 moxibustion groups and the 2 blank groups (P>0.05). There were no significant differences in the expression of CD, CD, and CD/CD ratio between the 2 moxibustion groups and the corresponding blank groups (P>0.05). Compared with the young blank group, the young moxibustion group had elevated IL-6 level (P<0.05); compared with the middle-aged blank group, the middle-aged moxibustion group had decreased IL-10 and IL-17 levels (P<0.05). Compared with the young blank group, the young moxibustion group exhibited increased Sobs index, Ace index, and Chao index (P<0.01, P<0.05), as well as increased relative abundance of Spirochaetota, Treponema, Turicibacter, Rikenellaceae_RC9_gut_group (P<0.05), and decreased relative abundance of Dubosiella (P<0.05). Compared with the middle-aged blank group, the middle-aged moxibustion group had increased relative abundance of Spirochaetota, Treponema, norank_f_Peptococcaceae (P<0.05), and decreased relative abundance of Proteobacteria, Allobaculum, and Faecalibaculum (P<0.05). Correlation analysis revealed that relative abundance of Eubacterium_xylanophilum_group and unclassified _f_Lachnospiraceae was negatively correlated with serum TNF-α level (r=-0.39, P=0.03; r=-0.24, P=0.04), while relative abundance of norank_f_norank_o_Clostridia_UCG-014 and Lactobacillus was positively correlated with serum TNF-α level (r=0.37, P=0.04; r=0.43, P=0.02). The relative abundance of Roseburia and Monoglobus was negatively correlated with serum IFN-γ level (r=-0.40, P=0.02; r=-0.44, P=0.01), while relative abundance of Lactobacillus was positively correlated with serum IL-10 level (r=0.43, P=0.02).@*CONCLUSION@#Moxibustion could improve immune function in healthy rats, and its mechanism may be related to the regulation of relative abundance of intestinal flora.

Ethanol extract of Herpetospermum caudigerum Wall ameliorates psoriasis-like skin inflammation and promotes degradation of keratinocyte-derived ICAM-1 and CXCL9 / 中西医结合学报

OBJECTIVE@#To explore whether the ethanol extract of Herpetospermum caudigerum Wall (EHC), a Xizang medicinal plant traditionally used for treating liver diseases, can improve imiquimod-induced psoriasis-like skin inflammation.@*METHODS@#Immunohistochemistry and immunofluorescence staining were used to determine the effects of topical EHC use in vivo on the skin pathology of imiquimod-induced psoriasis in mice. The protein levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-17A (IL-17A) in mouse skin samples were examined using immunohistochemical staining. In vitro, IFN-γ-induced HaCaT cells with or without EHC treatment were used to evaluate the expression of keratinocyte-derived intercellular cell adhesion molecule-1 (ICAM-1) and chemokine CXC ligand 9 (CXCL9) using Western blotting and reverse transcription-quantitative polymerase chain reaction. The protein synthesis inhibitor cycloheximide and proteasome inhibitor MG132 were utilized to validate the EHC-mediated mechanism underlying degradation of ICAM-1 and CXCL9.@*RESULTS@#EHC improved inflammation in the imiquimod-induced psoriasis mouse model and reduced the levels of IFN-γ, TNF-α, and IL-17A in psoriatic lesions. Treatment with EHC also suppressed ICAM-1 and CXCL9 in epidermal keratinocytes. Further mechanistic studies revealed that EHC suppressed keratinocyte-derived ICAM-1 and CXCL9 by promoting ubiquitin-proteasome-mediated protein degradation rather than transcriptional repression. Seven primary compounds including ehletianol C, dehydrodiconiferyl alcohol, herpetrione, herpetin, herpetotriol, herpetetrone and herpetetrol were identified from the EHC using ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry.@*CONCLUSION@#Topical application of EHC ameliorates psoriasis-like skin symptoms and improves the inflammation at the lesion sites. Please cite this article as: Zhong Y, Zhang BW, Li JT, Zeng X, Pei JX, Zhang YM, Yang YX, Li FL, Deng Y, Zhao Q. Ethanol extract of Herpetospermum caudigerum Wall ameliorates psoriasis-like skin inflammation and promotes degradation of keratinocyte-derived ICAM-1 and CXCL9. J Integr Med. 2023; 21(6): 584-592.

Expression level of cytokines in patients with sepsis and its effect on prognosis / 中华危重病急救医学

OBJECTIVE@#To observe the expression level of cytokines in patients with sepsis and its effect on prognosis.@*METHODS@#The clinical data of sepsis patients admitted to the intensive care unit (ICU) of the First Affiliated Hospital of Zhengzhou University from January 2020 to December 2022 were analyzed retrospectively, including gender, age, and acute physiology and chronic health evaluation II (APACHE II), blood routine, procalcitonin (PCT), C-reactive protein (CRP), and cytokines levels [interleukins (IL-2, IL-4, IL-6, IL-10, IL-17), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)] within 24 hours of admission to ICU. The 28-day prognosis of the patients was followed up. The patients were divided into survival group and death group according to the prognosis. The clinical data between the two groups of sepsis patients with different prognosis were compared. Binary Logistic regression analysis was used to analyze the independent risk factors affecting the prognosis of patients with sepsis, and the receiver operator characteristic curve (ROC curve) was drawn to evaluate the predictive value of each risk factor for the prognosis of patients with sepsis.@*RESULTS@#(1) A total of 227 patients with sepsis were enrolled, including 168 patients in the survival group (survival rate 74.0%) and 59 patients in the death group (mortality 26.0%). There were no significant differences in age (years old: 55.97±2.13 vs. 54.67±1.11) and gender (male: 71.2% vs. 57.1%) between the death group and the survival group (both P > 0.05), indicating that the baseline data of the two groups were comparable. (2) The APACHE II (19.37±0.99 vs. 14.88±0.61, P < 0.001) and PCT (μg/L: 12.39±2.94 vs. 4.14±0.90, P < 0.001) in the death group were significantly higher than those in the survival group, while the platelet count [PLT (×109/L): 144.75±12.50 vs. 215.99±11.26, P = 0.001] and thrombocytocrit [(0.14±0.01)% vs. (0.19±0.01)%, P = 0.001] were significantly lower than those in the survival group. (3) The level of IL-6 in the death group was significantly higher than that in the survival group (ng/L: 577.66±143.16 vs. 99.74±33.84, P < 0.001). There were no statistically significant differences in other cytokines, IL-2, IL-4, IL-10, TNF-α, IFN-γ and IL-17 between the death group and the survival group [IL-2 (ng/L): 2.44±0.38 vs. 2.63±0.27, P = 0.708; IL-4 (ng/L): 3.26±0.67 vs. 3.18±0.34, P = 0.913; IL-10 (ng/L): 33.22±5.13 vs. 39.43±2.85, P = 0.262; TNF-α (ng/L): 59.33±19.21 vs. 48.79±29.87, P = 0.839; IFN-γ (ng/L): 6.69±5.18 vs. 1.81±0.16, P = 0.100; IL-17 (ng/L): 2.05±0.29 vs. 2.58±0.33, P = 0.369]. (4) Binary Logistic regression analysis showed that APACHE II and IL-6 were independent risk factors affecting the prognosis of patients with sepsis [odds ratio (OR) and 95% confidence interval (95%CI) were 1.050 (1.008-1.093) and 1.001 (1.000-1.002), P values were 0.019 and 0.026, respectively]. (5) ROC curve analysis showed that APACHE II and IL-6 had certain predictive value for the prognosis of patients with sepsis, the area under the ROC curve (AUC) was 0.754 (95%CI was 0.681-0.827) and 0.592 (95%CI was 0.511-0.673), P values were < 0.001 and 0.035, respectively. When the optimal cut-off value of APACHE II was 16.50 score, the sensitivity was 72.6% and the specificity was 69.9%. When the optimal cut-off value of IL-6 was 27.87 ng/L, the sensitivity was 67.2% and the specificity was 52.8%.@*CONCLUSIONS@#APACHE II score and IL-6 level have certain predictive value for the prognosis of patients with sepsis, the higher APACHE II score and IL-6 level, the greater the probability of death in patients with sepsis.

A preliminary study on the role of V-domain Ig suppressor of T cell activation in juvenile idiopathic arthritis / 中国当代儿科杂志

OBJECTIVES@#To study the expression of V-domain Ig suppressor of T cell activation (VISTA) in peripheral blood of children with juvenile idiopathic arthritis (JIA) and its role in the pathogenesis of JIA.@*METHODS@#In this prospective study, peripheral blood was collected from 47 children with different subtypes of JIA and 10 healthy children. Flow cytometry was used to measure the expression levels of VISTA, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) on CD14+ mononuclear cells, CD4+ T lymphocytes, and CD8+ T lymphocytes.@*RESULTS@#The children with JIA had a significantly lower expression level of VISTA than the healthy children (P<0.05). There was a significant difference in the expression of VISTA between the children with different subtypes of JIA, with the lowest expression level in those with systemic JIA (P<0.05). There was also a significant difference in the expression of VISTA between different immune cells, with a significantly higher expression level on the surface of monocytes (P<0.05). Correlation analysis showed that VISTA was negatively correlated with the expression of IFN-γ and TNF-α on CD4+ T cells (r=-0.436 and -0.382 respectively, P<0.05), CD8+ T cells (r=-0.348 and -0.487 respectively, P<0.05), and CD14+ mononuclear cells (r=-0.582 and -0.603 respectively, P<0.05).@*CONCLUSIONS@#The insufficient expression of VISTA may be associated with the pathogenesis of JIA, and enhancing the immunomodulatory effect of VISTA might be one option for the treatment of JIA in the future.

Mechanism of Xiaoer Chiqiao Qingre Granules in clearing heat and removing food stagnation in suckling rats with fever and food accumulation based on metabolomics / 中国中药杂志

Children's fever is often accompanied by food accumulation. Traditional Chinese medicine believes that removing food stagnation while clearing heat of children can effectively avoid heat damage. To systematically evaluate the efficacy of Xiaoer Chiqiao Qingre Granules(XRCQ) in clearing heat and removing food accumulation and explore its potential mechanism, this study combined suckling SD rats fed with high-sugar and high-fat diet with injection of carrageenan to induce rat model of fever and food accumulation. This study provided references for the study on the pharmacodynamics and mechanism of XRCQ. The results showed that XRCQ effectively reduced the rectal temperature of suckling rats, improved the inflammatory environment such as the content of interleukin-1β(IL-1β), interleukin-2(IL-2), interferon-γ(IFN-γ), white blood cells, and monocytes. XRCQ also effectively repaired intestinal injury and enhanced intestinal propulsion function. According to the confirmation of its efficacy of clearing heat, the thermolytic mechanism of XRCQ was further explored by non-targeted and targeted metabolomics methods based on LTQ-Orbitrap MS/MS and UPLC-QQQ-MS/MS. Non-target metabolomics analysis of brain tissue samples was performed by QI software combined with SIMCA-P software, and 22 endogenous metabolites that could be significantly regulated were screened out. MetaboAnalyst pathway enrichment results showed that the intervention mechanism was mainly focused on tyrosine metabolism, tricarboxylic acid cycle, inositol phosphate metabolism, and other pathways. At the same time, the results of targeted metabolomics of brain tissue samples showed that XRCQ changed the vitality of digestive system, and inhibited abnormal energy metabolism and inflammatory response, playing a role in clearing heat and removing food stagnation from multiple levels.

Regulating the immune response to carbon tetrachloride-induced liver fibrosis in mice by blocking inducible co-stimulatory molecules and interleukin-33 / 中华肝脏病杂志

Objective: To investigate the effects of combined blockade of interleukin-33 (IL-33) and inducible co-stimulatory molecule (ICOS) on carbon tetrachloride-induced chronic liver fibrosis and imbalance of T helper lymphocyte subsets in mice. Methods: There were 40 BALB/c mice in each model and control group. Flow cytometry was used to determine the proportion of Th1/Th2/Th17 cells in the splenic lymphocyte suspension of mice, the expression levels of interferon γ, IL-4, and IL-17 in the splenic lymphocyte suspension of liver fibrosis mice after combined blockade of IL-33 and ICOS, and the pathological changes of liver histopathology in mice with liver fibrosis. Two independent sample t-test was used to compare data between groups. Results: Compared with the non-blocking group, the proportion of Th2 and Th17 cells in the IL-33/ICOS blocking group was significantly down-regulated (Th2: 65.96% ± 6.04% vs. 49.09% ± 7.03%; Th17: 19.17% ± 4.03% vs. 9.56% ± 2.03%), while the proportion of Th1 cells and Th1/Th2 ratio were up-regulated (Th1: 17.14% ± 3.02% vs. 31.93% ± 5.02%; Th1/Th2: 0.28 ± 0.06 vs. 0.62 ± 0.23), and the difference was statistically significant (t = 5.15, 6.03, 7.14, 4.28, respectively, with P < 0.05). After entering the chronic inflammation stage of liver fibrosis in mice (10 weeks), compared with the non-blocking group, the expression levels of IL-4 and IL-17 in the blockade group were significantly down-regulated [IL-4: (84.75 ± 14.35) pg/ ml vs. (77.88 ± 19.61) pg/ml; IL-17: (72.38 ± 15.13) pg/ml vs. (36.38 ± 8.65) pg/ml], while the expression of interferon γ was up-regulated [(37.25 ± 11.51) pg/ml vs. (77.88 ± 19.61) pg/ml], and the difference was statistically significant (t: IL-4: 4.71; IL-17: 5.84; interferon γ: 5.05, respectively, with P < 0.05). Liver histopathological results showed that hepatic necrosis, hepatic lobular structural disorder, and fibrous tissue hyperplasia were significantly lower in the blockade group than those in the non-blocking group at 13 weeks of liver fibrosis. Conclusion: Combined blockade of the ICOS signaling pathway and IL-33 can regulate Th2 and Th17 polarization, down-regulate the inflammatory response, and inhibit or prevent the occurrence and progression of fibrosis.

Dermatophagoides farinae induces conjunctival epithelial cell damage to promote neutrophil migration and neutrophil extracellular traps formation / 中国血吸虫病防治杂志

OBJECTIVE@#To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM).@*METHODS@#Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA.@*RESULTS@#Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells.@*CONCLUSIONS@#CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.

The role of natural killer cells in anti-infection and tumor therapy / 细胞与分子免疫学杂志

Natural killer (NK) cells are an important part of the body's innate immune system. As the first line of defense against pathogens, they need to be transformed into a mature state under the control of various cell signaling molecules and transcription factors to play cytotoxic and immune regulatory roles. Under the interaction of activated receptors and inhibitory receptors, NK cells are activated to perform a direct cell killing effect by secreting perforin and granzyme, or indirectly eliminate pathogenic microorganisms in the body by secreting various cytokines, such as type I and type II interferons. These functions of NK cells play a very important role in antiviral and anti-autoimmune diseases, especially in anti-tumor.

The number of TIGIT+CD8+ T cells increases but their cytokine secretion decreases in the lungs of Plasmodium yoelii infected mice / 细胞与分子免疫学杂志

Objective To investigate the effect of T cell immunoreceptor with Ig and ITIM domains (TIGIT) on the function of CD8+ T cells in the lungs of Plasmodium infected mice. Methods The lungs of the mice infected with Plasmodium yoelii were isolated, weighed and photographed after 12 days' infection. After dissolution, lung lymphocytes were isolated, counted and stained, and then the contents of CD8+ and TIGIT+CD8+ T cells were detected by flow cytometry. The expressions of L selectin (CD62L), CD69, programmed death 1 (PD-1), CD25, and C-X3-C motif chemokine receptor 1 (CX3CR1) on TIGIT+CD8+ T cells were detected by flow cytometry. After stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, the ability of TIGIT+CD8+T cells to secrete interferon γ(IFN-γ), interleukin 21 (IL-21), IL-4, IL-17, and IL-10 was detected. Results The body mass of mice with Plasmodium infection was reduced. The lungs became darker, and the ratio of the lung mass to body mass was significantly increased. Compared with the normal mice, the percentages and absolute quantity of CD8+ and TIGIT+CD8+ T cells in the lungs of the infected mice were significantly increased. The percentage of TIGIT+CD8+ T cells expressing CD62L in the infected group was significantly lower, while the percentage of the CD69, PD-1, and CX3CR1 cells were significantly higher than that of TIGIT+CD8+ T cells from the normal mice. The percentages of TIGIT+CD8+ T cells secreting IL-21, IL-4, IL-17 and IL-10 cells in the infected group were significantly lower. Conclusion The lung lesions from mice with Plasmodium infection are obvious, the numbers of TIGIT+CD8+ T cells increase, and these cells express a variety of activation-related molecules, but the ability to secrete cytokines is reduced.

Mechanism of regulatory T cells in heat stroke-induced acute kidney injury / 中华危重病急救医学

OBJECTIVE@#To investigate the mechanism of regulatory T cells (Treg) in heat stroke (HS)-induced acute kidney injury (AKI).@*METHODS@#Male SPF Balb/c mice were randomly divided into control group, HS group (HS+Rat IgG), HS+PC61 group, and HS+Treg group (n = 6). The HS mice model was established by making the body temperature of the mice reach 42.7 centigrade at room temperature 39.5 centigrade with relative humidity 60% for 1 hour. In HS+PC61 group, 100 μg PC61 antibody (anti-CD25) was injected through the tail vein in consecutive 2 days before the model was established to eliminate Tregs. Mice in HS+Treg group was injected with 1×106 Treg via tail vein immediately after successful modeling. The proportion of Treg infiltrated in the kidney, serum creatinine (SCr) and histopathology, levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) both in the serum and kidney tissue, as well as proportion of neutrophils and macrophages located in the kidney were observed at 24 hours after HS.@*RESULTS@#HS dampened renal function and exaggerated kidney injury, up-regulated levels of inflammatory cytokines both in local kidney and circulation, and increased infiltration of neutrophils and macrophages to the injured kidneys. The proportion of Treg (Treg/CD4+) infiltrated in kidney was significantly decreased in HS group, compared with control group [(3.40±0.46)% vs. (7.67±0.82)%, P < 0.01]. Compared with HS group, local Tregs in kidney were almost completely depleted via PC61 antibody [(0.77±0.12)% vs. (3.40±0.46)%, P < 0.01]. Depletion of Tregs could exacerbate HS-AKI, indicating by increased serum creatinine [SCr (mmol/L): 348.22±35.36 vs. 254.42±27.40, P < 0.01] and pathological injury (Paller score: 4.70±0.20 vs. 3.60±0.20, P < 0.01), incremental levels of IFN-γand TNF-α both in injured kidney and serum [serum IFN-γ (ng/L): 747.70±64.52 vs. 508.46±44.79, serum TNF-α (ng/L): 647.41±26.62 vs. 464.53±41.80, both P < 0.01], and more infiltrated neutrophils and macrophages in the injured kidney [neutrophil proportion: (6.63±0.67)% vs. (4.37±0.43)%, macrophage proportion: (38.70±1.66)% vs. (33.19±1.55)%, both P < 0.01]. On the contrast, adoptive transfer of Tregs could reverse the aforementioned effects of Treg depletion, indicating by incremental proportion of Tregs in the injured kidney [(10.58±1.19)% vs. (3.40±0.46)%, P < 0.01], decreased serum creatinine [SCr (mmol/L): 168.24±40.56 vs. 254.42±27.40, P < 0.01] and pathological injury (Paller score: 2.73±0.11 vs. 3.60±0.20, P < 0.01), reduced levels of IFN-γ and TNF-α both in injured kidney and serum [serum IFN-γ (ng/L): 262.62±22.68 vs. 508.46±44.79, serum TNF-α (ng/L): 206.41±22.58 vs. 464.53±41.80, both P < 0.01], and less infiltrated neutrophils and macrophages in the injured kidney [neutrophil proportion: (3.04±0.33)% vs. (4.37±0.43)%, macrophage proportion: (25.68±1.93)% vs. (33.19±1.55)%, both P < 0.01].@*CONCLUSIONS@#Treg might be involved in HS-AKI, possibly via down-regulation of pro-inflammatory cytokines and infiltration of inflammatory cells.

Down-regulation of DNA key protein-FEN1 inhibits OSCC growth by affecting immunosuppressive phenotypes via IFN-γ/JAK/STAT-1 / 国际口腔科学杂志·英文版

Oral squamous cell carcinoma (OSCC) escape from the immune system is mediated through several immunosuppressive phenotypes that are critical to the initiation and progression of tumors. As a hallmark of cancer, DNA damage repair is closely related to changes in the immunophenotypes of tumor cells. Although flap endonuclease-1 (FEN1), a pivotal DNA-related enzyme is involved in DNA base excision repair to maintain the stability of the cell genome, the correlation between FEN1 and tumor immunity has been unexplored. In the current study, by analyzing the clinicopathological characteristics of FEN1, we demonstrated that FEN1 overexpressed and that an inhibitory immune microenvironment was established in OSCC. In addition, we found that downregulating FEN1 inhibited the growth of OSCC tumors. In vitro studies provided evidence that FEN1 knockdown inhibited the biological behaviors of OSCC and caused DNA damage. Performing multiplex immunohistochemistry (mIHC), we directly observed that the acquisition of critical immunosuppressive phenotypes was correlated with the expression of FEN1. More importantly, FEN1 directly or indirectly regulated two typical immunosuppressive phenotype-related proteins human leukocyte antigen (HLA-DR) and programmed death receptor ligand 1 (PD-L1), through the interferon-gamma (IFN-γ)/janus kinase (JAK)/signal transducer and activator transcription 1 (STAT1) pathway. Our study highlights a new perspective on FEN1 action for the first time, providing theoretical evidence that it may be a potential immunotherapy target for OSCC.

Grape Seed Extract Attenuates Demyelination in Experimental Autoimmune Encephalomyelitis Mice by Inhibiting Inflammatory Response of Immune Cells / 中国结合医学杂志

OBJECTIVE@#To examine the anti-inflammatory effect of grape seed extract (GSE) in animal and cellular models and explore its mechanism of action.@*METHODS@#This study determined the inhibitory effect of GSE on macrophage inflammation and Th1 and Th17 polarization in vitro. Based on the in vitro results, the effects and mechanisms of GSE on multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE) mice model were further explored. The C57BL/6 mice were intragastrically administered with 50 mg/kg of GSE once a day from the 3rd day to the 27th day after immunization. The activation of microglia, the polarization of Th1 and Th17 and the inflammatory factors such as tumor necrosis factor- α (TNF- α), interleukin-1 β (IL-1 β), IL-6, IL-12, IL-17 and interferon-γ (IFN-γ) secreted by them were detected in vitro and in vivo by flow cytometry, enzyme linked immunosorbent assay (ELISA), immunofluorescence staining and Western blot, respectively.@*RESULTS@#GSE reduced the secretion of TNF-α, IL-1 β and IL-6 in bone marrow-derived macrophages stimulated by lipopolysaccharide (P<0.01), inhibited the secretion of TNF-α, IL-1 β, IL-6, IL-12, IL-17 and IFN-γ in spleen cells of EAE mice immunized for 9 days (P<0.05 or P<0.01), and reduced the differentiation of Th1 and Th17 mediated by CD3 and CD28 factors (P<0.01). GSE significantly improved the clinical symptoms of EAE mice, and inhibited spinal cord demyelination and inflammatory cell infiltration. Peripherally, GSE downregulated the expression of toll-like-receptor 4 (TLR4) and Rho-associated kinase (ROCKII, P<0.05 or P<0.01), and inhibited the secretion of inflammatory factors (P<0.01 or P<0.05). In the central nervous system, GSE inhibited the infiltration of CD45+CD11b+ and CD45+CD4+ cells, and weakened the differentiation of Th1 and Th17 (P<0.05). Moreover, it reduced the secretion of inflammatory factors (P<0.01), and prevented the activation of microglia (P<0.05).@*CONCLUSION@#GSE had a beneficial effect on the pathogenesis and progression of EAE by inhibiting inflammatory response as a potential drug and strategy for the treatment of MS.

Efecto del estrés crónico y de la masticación sobre los niveles séricos de interleuquina-6 e interferón-gama / Effect of chronic stress and chewing on serum interleukin-6 and interferon-gamma levels

Introducción: el estrés crónico afecta el equilibrio inmunológico alterando los niveles séricos de interleuquina-6 (IL-6) e interferón gama (INF-γ), dicha alteración afecta al sistema nervioso y al comportamiento humano. La masticación adecuada disminuiría dichos efectos. El objetivo del estudio fue determinar el efecto del estrés crónico y de la masticación sobre los niveles séricos de IL-6 e INF-γ. Métodos: experimento donde se emplearon 64 ratones Balb/c de 8 semanas de edad. Se dividieron en 4 tratamientos: Grupo NE: Masticación normal + estrés, Grupo N: masticación normal sin estrés, Grupo DE: Masticación deficiente + estrés, Grupo D: masticación deficiente sin estrés. Mediante test de ELISA se midió IL-6 e IFN-γ alfinal de la 4ta y de la 8va semana de tratamiento. Resultados: tanto la IL-6 como el IFN-γ fueron mayores en el grupo DE (p<0,05) al final de la 4ta semana. Al evaluarlos al término de la 8va semana se observó que en el grupo NE se incrementó la IL-6 respecto al resto de grupos (p<0,0001), y en el grupo DE fue donde se encontró mayor cantidad de IFN-γ (p<0,0001). Conclusión: el estrés crónico y la masticación deficiente incrementan los niveles séricos de IL-6 e IFN-γ. En cambio, la adecuada masticación disminuye el nivel de tales citoquinas al final de la cuarta semana de tratamiento.

Introduction: chronic stress affects the immune balance by altering the serum levels of interleukin-6 (IL-6) and gamma interferon (INF-γ), this alteration affects the nervous system and human behavior. Appropriate chewing would lessen these effects. The aim of this study was to determine the effect of chewing and chronic stress over serum levels of IL-6 and INF-γ. Methods: experiment in which 64 Balb/C mice of 8 weeks of age were used, they were divided into 4 treatments: Group NE: Normal chewing + stress, Group N: normal chewing without stress, Group DE: Chewing poor + stress, Group D: poor chewing without stress. IL-6 and IFN-γ were measured by ELISA after 4 and 8 weeks of treatment. Results: both IL-6 and IFN-γ were higher in the DE group (p < 0,05) at the end of fourth week of treatment. When evaluating the animals at the end of the eighth week of treatment, it was observed that in the NE group, the IL-6 was increased with respect to the rest (p < 0,0001) and the DE group showed more IFN-γ (p < 0,0001). Conclusion: stress and poor chewing increase serum IL-6 and IFN-γ. In contrast, appropriate chewing decreases the effects of stress on the increase of such cytokines at the end of the fourth week of treatment in animals.

Therapeutic effects of the extract of Sancao Formula, a Chinese herbal compound, on imiquimod-induced psoriasis via cysteine-rich protein 61 / 中西医结合学报

OBJECTIVE@#Psoriasis is a common chronic inflammatory skin disease that is prone to recurrence, and the proinflammatory factor, cysteine-rich protein 61 (Cyr61), is important in its pathophysiology. Long-term clinical practice has shown that Sancao Formula (SC), a Chinese herbal compound, is effective in the treatment of psoriasis, but the precise mechanism remains unknown. In this study, we investigate the mechanism by which SC extract alleviates imiquimod (IMQ)-induced psoriasis.@*METHODS@#The expression of Cyr61 in psoriatic lesions and normal healthy skin was detected using immunohistochemical analysis to investigate the biological role of Cyr61 in models of psoriatic inflammation. A psoriatic mouse model was established by topical application of IMQ, and the effect of topical application of SC extract was evaluated using the psoriasis area and severity index (PASI) score, hematoxylin-eosin staining, and histopathological features of the skin. Next, a HaCaT cell inflammation model was established using interferon-γ (IFN-γ), and the effect of SC extract on the mRNA and protein levels of Cyr61 and intercellular cell adhesion molecule-1 (ICAM-1) was confirmed using Western blot and quantitative real-time polymerase chain reaction analyses.@*RESULTS@#Immunohistochemical staining showed that the expression of Cyr61 in psoriatic lesions was higher than that in normal skin samples (78.26% vs 41.18%, P < 0.05), and the number of Cyr61-positive cells in psoriatic lesions was also significantly higher than in normal skin (18.66 ± 2.51 vs 4.33 ± 1.52, P < 0.05). Treatment in mice with IMQ-induced psoriasis showed that SC extract could significantly improve the inflammatory phenotype, PASI score (10.875 ± 0.744 vs 3.875 ± 0.582, P < 0.05), and pathological features compared with those in IMQ model group; SC treatment was also associated with decreased levels of Cyr61 and ICAM-1. In the IFN-γ-induced inflammatory cell model, the mRNA and protein levels of Cyr61 and ICAM-1 were upregulated, while the SC extract downregulated the levels of Cyr61 and ICAM-1.@*CONCLUSION@#The results provide a theoretical basis for the involvement of Cyr61 in the pathogenesis of psoriasis, and suggest that SC should be used to target Cyr61 for the prevention of psoriasis recurrence.

Acupoint Autohemotherapy Attenuates Atopic Dermatitis Lesions by Regulating Th1/Th2 Balance in DNCB-Induced BALB/c Mice / 中国结合医学杂志

OBJECTIVE@#To evaluate the therapeutic effects of acupoint autohemotherapy (A-AHT) on 1-chloro-2,4-dinitrobenzene (DNCB)-induced atopic dermatitis (AD) in mice focusing on regulating T helper 1/T helper 2 (Th1/Th2) immune responses.@*METHODS@#Thirty BALB/c mice were divided into 5 groups by a random number table, including normal control (NC), AD model (AD), A-AHT, sham A-AHT (sA-AHT), and acupoint injection of normal saline (A-NS) groups, 6 mice per group. Mice were challenged by DNCB for the establishment of experimental AD model. On the 8th day, except for the NC and AD groups, the mice in the other groups received management once every other day for a total of 28 days. For the A-AHT and sA-AHT groups, 0.05 mL of autologous whole blood (AWB) was injected into bilateral Zusanli (ST 36) and Quchi (LI 11) and sham-acupoints (5 mm lateral to ST 36 and LI 11), respectively. The A-NS group was administrated with 0.05 mL of normal saline by acupoint injection into ST 36 and LI 11. Dermatitis severity for dorsal skin of mice was determined using the Severity Scoring of Atopic Dermatitis (SCORAD) every week. The total immunoglobulin E (IgE), interleukin-4 (IL-4), and interferon-gamma (IFN-γ) cytokine levels in serum were examined by enzyme-linked immunosorbent assay (ELISA). Spleen Th1/Th2 expression were analyzed via flow cytometry and immunohistochemical assay was used to detect T-box expressed in T cell (T-bet) and GATA-binding protein 3 (GATA3) expressions in skin lesions of mice.@*RESULTS@#Compared with the AD group, both A-AHT and sA-AHT reduced the SCORAD index and serum IgE level (P<0.05 or P<0.01); A-AHT, sA-AHT and A-NS down-regulated serum IL-4 level and upregulated IFN-γ/IL-4 ratio (P<0.05 or P<0.01); A-AHT regulated the Th1/Th2 shift specifically and increased the related transcription factors such as T-bet expression and T-bet/GATA3 ratio (P<0.05).@*CONCLUSION@#A-AHT showed significant effectiveness on the AD model mice, through regulating Th1/Th2 immune responses.

Acupuncture combined with western medication for ocular myasthenia gravis: a randomized controlled trial / 中国针灸

OBJECTIVE@#To compare the clinical efficacy between acupuncture combined with western medication and simple western medication for ocular myasthenia gravis (OMG), and to explore its possible mechanism.@*METHODS@#A total of 60 patients of ocular myasthenia gravis were randomized into an acupuncture combined with western medication group (30 cases, 1 case dropped off) and a western medication group (30 cases, 2 cases dropped off). Oral pyridostigmine bromide tablet and prednisone acetate tablet were given in the western medication group. On the basis of the treatment in the western medication group, Tongdu Tiaoqi acupuncture (acupuncture for unblocking the governor vessel and regulating qi ) was applied at Baihui (GV 20), Fengfu (GV 16), Hegu (LI 4), Zusanli (ST 36), etc. in the acupuncture combined with western medication group, once a day, 6 days a week. The treatment was given 8 weeks in both groups. Before and after treatment, the OMG clinical absolute score was observed, electrophysiological indexes of orbicularis oculi (value of mean jitter, percentage of jitter >55 μs and percentage of blocks) were measured by single-fiber electromyography (SFEMG), serum levels of acetylcholine receptor antibody (AChR-Ab), interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were detected by ELISA method.@*RESULTS@#After treatment, the OMG clinical absolute scores, values of mean jitter, percentages of jitter >55 μs, percentages of blocks and serum levels of AChR-Ab, IFN-γ and IL-4 were decreased compared before treatment in both groups (P<0.05), and those in the acupuncture combined with western medication group were lower than the western medication group (P<0.05).@*CONCLUSION@#Acupuncture combined with western medication can effectively improve ptosis, palpebra superior fatigability, eye movement disorder and neuromuscular junction dysfunction in patients with ocular myasthenia gravis, the therapeutic effect is superior to simple western medication. Its mechanism may be related to down-regulating serum levels of AChR-Ab, IFN-γ and IL-4 and promoting the recovery of orbicularis oculi function.