Effects of CD68 + CD163 + M2 macrophages on tumor angiogenesis in hepatocellular carcinoma / 中华微生物学和免疫学杂志

Objective:To study the effects of CD68 + CD163 + M2 macrophages on promoting tumor angiogenesis in hepatocellular carcinoma. Methods:CD68 + M0 macrophages in human spleen tissues were separated by mechanical grinding and magnetic bead separation and used to induce CD68 + CD163 + M2 macrophages in vitro with the presence of IL-4 and IL-13. CD68 + M0 and CD68 + CD163 + M2 macrophages were respectively co-cultured with human umbilical vein endothelial cells (HUVEC) in vitro. CCK-8 assay and scratch test were performed to detect the viability and migration ability of HUVEC. Tube formation assay was used to analyze in vitro angiogenesis. Expression of vascular endothelial growth factor receptor 2 (VEGFR2), Notch1 and Dll4 in HUVEC was detected by Western blot. Enzyme linked immunosorbent assay (ELISA) was used to detect the expression of vascular endothelial growth factor (VEGF) and IL-8 in media. BALB/c nude mice were used to construct HepG2 hepatoma model. CD68 + M0 and CD68 + CD163 + M2 macrophages were respectively injected into the mice. Expression of CD105 in tumor tissues was detected by immunohistochemistry. Expression of VEGF, VEGFR2, Notch1 and Dll4 in tumor tissues was detected by Western blot. Results:(1) IL-4 and IL-13 could induce CD68 + M0 macrophages to differentiate into CD68 + CD163 + M2 macrophages. (2) In the cell experiments, CD68 + M0 macrophages did not significantly promote the angiogenesis of HUVEC, and there was no significant change in the level of VEGF, VEGFR2, Notch1, Dll4 or IL-8. CD68 + CD163 + M2 macrophages could promote the in vitro tube formation of HUVEC, which was related to the activation of VEGF-VEGFR2-Notch1/Dll4 signaling pathway. (3) In the animal experiments, CD68 + CD163 + M2 macrophages could promote the expression of CD105, the formation of tumor vessels and the expression of VEGF-VEGFR2-Notch1/Dll4 signals. Conclusions:CD68 + CD163 + M2 macrophages could promote the formation of tumor vessels in hepatocellular carcinoma by secreting angiogenic factors including VEGF and activating VEGF-VEGFR2-Notch1/Dll4 signals.

Role of HIF-1α in hydrogen-induced inhibition of lipopolysaccharide-induced inflammatory responses in mouse macrophages / 中华麻醉学杂志

Objective:To evaluate the role of hypoxia-inducible factor-1α (HIF-1α) in hydrogen-induced inhibition of lipopolysaccharide (LPS)-induced inflammatory responses in mouse macrophages.Methods:The mouse RAW264.7 macrophages cultured in vitro were divided into 4 groups ( n=24 each) according to the random number table method: control group (C group), LPS group (L group), hydrogen-rich solution plus LPS group (H+ L group), and hydrogen-rich solution plus LPS plus HIF-1α inhibitor 2-methoxyestradiol (2ME2) group (H+ L+ M group). LPS 1 μg/ml was added, and the cells were incubated for 6 h in group L. In group L+ H, LPS was added first, the medium was changed to 0.6 mmol/L hydrogen-rich solution, and cells were incubated for 6 h. In group H+ L+ M, 2ME2 10 μmol/L was given first, cells were then incubated for 30 min, LPS and hydrogen-rich solution were added, and cells were incubated for 6 h. Western blot was used to determine the expression of HIF-1α, Beclin-1, Bcl-2/E1B-19 kDa interacting protein 3 (BNIP3) and LC3.Enzyme-linked immunosorbent assay was used to detect the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in the supernatant.The number of autophagosomes was observed using a transmission electron microscope. Results:Compared with group C, the concentrations of TNF-α, IL-6 and IL-1β in the supernatant were significantly increased, the expression of HIF-1α, Beclinl and BNIP3 in macrophages was up-regulated, the ratio of LC3Ⅱ/LC3Ⅰ was increased, and the number of autophagosomes was increased in group L ( P<0.05). Compared with group L, the concentrations of TNF-α, IL-6 and IL-1β were significantly decreased, the expression of HIF-1α, Beclin-1 and BNIP3 in macrophages was up-regulated, LC3Ⅱ/LC3Ⅰ ratio was increased, and the number of autophagosomes was increased in group H+ L ( P<0.05). Compared with group H+ L, the concentrations of TNF-α, IL-6 and IL-1β in the supernatant were significantly decreased, the expression of HIF-1α, Beclin-1, and BNIP3 in macrophages was down-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was decreased, and the number of autophagosomes was decreased in group H+ L+ M ( P<0.05). Conclusion:HIF-1α-mediated activation of autophagy is involved in the process of hydrogen-induced inhibition of LPS-induced inflammatory responses in mouse macrophages.

Effects of ozone on iron death in spinal cord neurons of rats with neuropathic pain / 国际生物医学工程杂志

Objective:To investigate whether ferroptosis of nerve cells exists in a rat model of neuropathic pain (NP), and to explore the mechanism of O 3 treatment of NP. Methods:Sixty male SD rats were randomly divided into three groups: neuropathic pain model group (NP group), sham operation group (Sham group) and ozone group (O 3 group). The partial sciatic nerve ligation was used in the NP and O 3 groups to construct a neuropathic pain model. The Sham group was subjected to sham surgery. In the O 3 group, 15 μl of O 3 (40 μg/ml) was injected locally at the injury site, and the NP and Sham were injected with the same amount of air, once per day. The mechanical contraction response threshold (MWT) and thermal contraction latency (TWL) of the rats were measured 1 day before the surgery (T0) and 1, 3, 7, 14 days after the surgery (T1 to T4). The spinal cord segments of rats were collected. The expression levels of glutathione peroxidase 4 (GPX4) and long chain fatty acid coenzyme A synthetase 4 (ACSL4) at T1 to T4 was detected using Western Blot. The number of NeuN+ neuron cells in the spinal dorsal horn at T4 was detected using immunofluorescence technology. The specific changes of ferroptosis at T4 was observed by a transmission electron microscopy. Iron deposition in the spinal dorsal horn at T1 to T4 was measured using ferroptosis kits. Results:Compared with the Sham group, rats in the NP group and O 3 group showed decreasing of MWT decreased and shortening of TWL at T2 to T4, decreasing of NeuN+ neurons in spinal dorsal horn at T4, specific changes of ferroptosis in mitochondria at T4, and increasing of iron content in nerve tissue at T2 to T4. Compared with the Sham group, rats in NP group showed decreasing of GPX4 level and increasing of ACSL4 level. Compared with the NP group, rats in the O 3 group showed increasing of MWT and prolonging of TWL at T2 to T4, increasing of the GPX4 level and decreasing of ACSL4 level at T2 to T4, increasing of the number of NeuN+ neuron cells in the spinal dorsal horn, improving of the mitochondrial atrophy of nerve cells, and decreasing of the iron content in nerve tissue at T2 to T4. The above results are statistically significant (all P<0.05). Conclusions:The mechanism of O 3 in treating neuropathic pain may be through inhibition of iron death.

An investigation of the influencing factors for neck and shoulder pain among dentists in several grade A tertiary hospitals in Tianjin, China / 中华劳动卫生职业病杂志

Objective@#To investigate the incidence of neck and shoulder pain among dentists in several hospitals in Tianjin, China and the main factors for the onset of neck and shoulder pain, and to provide ideas for reducing the incidence rate of neck and shoulder pain in dentists.@*Methods@#In December 2018, a total of 140 dentists from General Hospital of Tianjin Medical University, The Second Hospital of Tianjin Medical University, and Tianjin Stomatological Hospital were selected as respondents. A self-designed questionnaire was used to investigate the incidence rate of neck and shoulder pain among the dentists and related influencing factors. A total of 140 questionnaires were distributed, among which 129 (96.9%) were usable questionnaires. The questionnaire contained the questions on personal information and conditions of neck and shoulder pain. The continuous data were expressed as Mean±SD, and the categorical data were expressed as percentage (%) . The multivariate logistic regression analysis was used to investigate influencing factors.@*Results@#Among the 129 respondents, 120 (93.02%) had neck and shoulder pain. The prevalence rates of neck pain alone, shoulder pain alone, and neck and shoulder pain were 34.9% (45/129) , 31.8% (41/129) , and 26.3% (34/129) , respectively. Certain factors, such as age, sex, exercise, working time, bad sitting posture, inappropriate seat, and engagement in periodontology or orthodontics, had a linear relationship with the incidence rate of neck and shoulder pain among the respondents (P<0.05) .@*Conclusion@#There is a high incidence rate of neck and shoulder pain among the dentists in some hospitals in Tianjin. Dentists should be encouraged to control the number of consultations, adjust the sitting posture during work, arrange working hours reasonably, and strengthen physical exercise, so as to reduce the incidence rate of neck and shoulder pain.

Interleukin-17-mediated inflammation promotes nonalcoholic fatty liver disease in mice with regulation of M1-type macrophage polarization / 中华肝脏病杂志

Objective@#To study the mechanism of interleukin (IL)-17 in mice with non-alcoholic fatty liver disease for promoting M1-type macrophage polarization to exacerbate liver inflammation, and to provide references for the mechanism of NAFLD occurrence and development.@*Methods@#A mouse model of NAFLD was constructed by high-fat diet. Mice were divided into control group, model group, IL-17 group, and anti IL-17 group. Histopathological changes of the liver were observed by HE staining. The serum levels of ALT and AST in peripheral blood of mice was detected by chemical colorimetry. Macrophages labeled with F4/80-PE, CD11C-FITC was designated as M1-type macrophages, those labeled with F4/80-PE, and CD206-APC was designated as M2-type macrophages. The proportion of M1 and M2 macrophages infiltrated into the liver tissues of mice were measured by flow cytometry. CD168 expression level of liver tissues was detected using immunohistochemistry. Protein and mRNA levels of the marker molecules (iNOS, TNF-alpha and IL-6) of M1 macrophages were detected using ELISA and RT-Q PCR. Western blot was used to detect the protein expression of JAK-STAT signal pathway and the expression level of MCP-1. Data were analyzed using one-way ANOVA and t-test.@*Results@#High-fat diet NAFLD mice model was successfully constructed. IL-17 had increased the proportion of M1 macrophages in mice liver tissues and decreased the proportion of M2 macrophages (P < 0.05). The proportion of M1 and M2 macrophages in the liver tissues of normal mice was 7.9% ± 1.1% and 19.2% ± 1.8%. The proportion of M1 and M2 macrophages in the model group was 17.3% ± 2.5% and 15.0% ± 2.1. The proportion of M1 macrophages (33.8% ± 4.2%) in IL-17 group was higher than model group, while the proportion of M2 macrophages (7.8% + 1.0%) in IL-17 group was lower than model group. Protein and mRNA marker levels of M1 macrophage (iNOS, IL-12, TNFα and IL-6) in liver tissues were significantly higher than model group, control group, and anti-IL-17 group (P < 0.05). The expression levels of JAK1, STAT1, MCP-1, and CD168 in mice liver tissues of IL-17 group had increased (P < 0.05). The levels of aspartate and alanine aminotransferases in peripheral blood of mice in IL-17 group were significantly higher than other three groups (P < 0.05).@*Conclusion@#IL-17 can promote M1-type macrophage polarization, and exacerbates the liver inflammatory response to accelerate the progression of NAFLD in mice.

Effects of dl-3-n-butylphthalide on angiogenesis of human umbilical vein endothelial cells under ischemia/hypoxia condition:in vitro experiment and its mechanism / 中华神经科杂志

Objective To investigate the effects of dl-3-n-butylphthalide ( NBP) on angiogenesis of human umbilical vein endothelial cells ( HUVEC) in vitro under ischemia/hypoxia and the correlation with vascular endothelial growth factor/vascular endothelial growth factor receptor 2 ( VEGF/VEGFR2 )-Notch1/Dll4 signaling pathway .Methods The control group , ischemia/hypoxia group , and ischemia/hypoxia +NBP group ( high dose group and low dose group ) were set up after HUVEC subculture . The cell concentration was adjusted to be 1 ×105/ml, and each group was randomly added 1 ml (1 ×105 cells in each group).Both of the ischemia/hypoxia group and the ischemia/hypoxia +NBP group were cultured under the condition of ischemia and hypoxia .The NBP concentrations of the high and low dose groups were 20 μmol/L and 5 μmol/L respectively.The cell viability of each group was detected by cell counting kit-8, and cell scratch test was used to detect the migration ability of the cells in each group .The cell formation ability of each group was examined by in vitro angiogenesis assay.Western blotting was used to detect the expressions of receptor proteins VEGFR2, Notch1 and Dll4,and mRNA expressions of VEGF, VEGFR2, Notch1 and Dll4 were detected by real-time quantitative PCR.Results NBP increased the survival rates of HUVEC under ischemia/hypoxia condition.In the low dose group, the survival rates of HUVEC at 6, 12, 24, 48 h were 78.6％ ±3.0％, 59.6％ ±5.3％, 44.6％ ±4.2％, 38.2％ ±4.3％, respectively, significantly higher than that in the ischemia/hypoxia group (75.2％±5.8％, 53.2％±4.8％, 36.2％± 7.8％, 22.5％±4.1％;t=4.513, 6.231, 9.322, 9.674; P=0.021, 0.018, 0.026, 0.015).In the high dose group, the survival rates of HUVEC at 6, 12, 24, 48 h were 88.6％±6.3％, 67.5％±5.4％, 53.3％±4.2％, 46.3％±3.9％, respectively , also significantly higher than that in the ischemia/hypoxia group (t=8.123, 11.211, 12.312, 14.154;P=0.001, 0.002, 0.001, 0.001).The mobility of the low dose group was 52.3％+4.2％, with statistically significant difference compared with that in the ischemia /hypoxia group ( 18.5％ ±3.2％) and the control group ( 22.3％ ±4.1％; t=18.324, 15.183; P=0.000, 0.000).The mobility of the high dose group was 87.5％±5.2％, also with statistically significant difference compared with that in the ischemia/hypoxia group and the control group ( t=22.142, 19.341;P=0.000, 0.000).NBP increased the protein and mRNA expression of VEGF , VEGFR2, Notch1 and Dll4.The relative expression of VEGFR2, Notch1 and Dll4 in the low dose group was 1.12 ±0.17, 0.35 ± 0.07 and 0.42 ±0.08, respectively, with statistically significant difference compared with that in the ischemia/hypoxia group (0.82 ±0.05, 0.30 ±0.03, 0.32 ±0.04;t=6.120, 2.123, 4.112;P=0.000, 0.020, 0.003).The relative expression of VEGFR2, Notch1 and Dll4 in the high dose group was 1.30 ± 0.15, 0.41 ±0.10 and 0.48 ±0.11, respectively, also with statistically significant difference compared with that in the ischemia/hypoxia group ( t=8.122, 3.851, 5.130; P=0.000, 0.000, 0.001 ) .The mRNA expressions of VEGF , VEGFR2, Notch1 and Dll4 in the low dose group were 0.43 ±0.08, 0.41 ± 0.05, 0.38 ±0.03 and 0.36 ±0.04, respectively, with statistically significant difference compared with that in the ischemia/hypoxia group (0.28 ±0.03, 0.34 ±0.04, 0.27 ±0.03, 0.19 ±0.04;t=3.122, 3.825, 4.311, 5.211; P=0.000, 0.006, 0.001, 0.000).And the mRNA expressions of VEGF, VEGFR2, Notch1 and Dll4 in the high dose group were 0.58 ±0.05, 0.50 ±0.06, 0.41 ±0.05, 0.52 ±0.06, respectively, also with statistically significant difference compared with that in the ischemia /hypoxia group (t=4.225, 4.872, 5.311, 8.220;P=0.000, 0.000, 0.000, 0.000).Conclusions NBP can promote HUVEC to form blood vessels under ischemia/hypoxia condition , the mechanism of which may be related to the activation of VEGF/VEGFR2-Notch1/Dll4 signaling pathway .It may be one of the mechanisms that NBP improves cerebral microcirculation in acute ischemic stroke .

Helper T cell Th9 and its cytokine interleukin-9 in peripheral blood of patients with Parkinson's disease / 中华神经科杂志

Objective To investigate the proportion of helper T cell subset Th 9 and the expression of its cytokine interleukin-9(IL-9)in Parkinson's disease(PD)and the clinical significance.Methods Seventy-two patients diagnosed with PD between January 2016 and June 2017 and 20 healthy volunteers in the same period were selected.The PD patients were staged according to the Hoehn-Yahr(H-Y)staging method,21 in stage one,19 in stage two,18 in stage three,11 in stage four,and three in stage five.The proportion of Th9 subset in peripheral blood of PD patients and healthy volunteers was measured by flow cytometry.The expression of IL-9 in peripheral blood of PD patients and healthy volunteers was measured by enzyme-linked immunosorbent assay.Results The proportion of Th9 cells in peripheral blood of PD patients(1.27%±0.34%)was significantly higher than that of healthy volunteers(0.61%±0.11%,t=8.530,P<0.05),and the higher stage of PD,the higher proportion of Th9,suggesting that the proportion of Th9 was related to the PD staging.IL-9 was also highly expressed in PD patients((16.04 ±2.94) pg/ml)and had statistically significant difference compared with healthy volunteers((7.53 ±0.70)pg/ml;t=12.781,P<0.05).IL-9 was similar to Th9, the higher stage of PD, the higher expression of IL-9. Conclusion The proportion of Th9 cells and the expression of IL-9 in peripheral blood of patients with PD increased significantly,having a significant relationship with the H-Y staging of PD.

To study the correlation of peripheral blood F-box and WD repeat domain-containing domain protein7 and myeloid cell leukemia-1 expression levels with the prognosis of Icotinib administration in elderly patients with advanced non-small-cell lung cancer / 中华老年医学杂志

Objective To study the correlation of the prognosis of Icotinib administration with the expression levels of F-box and WD repeat domain-containing 7(FBW7) and myeloid cell leukemia-1 (MCL-1) in peripheral blood in elderly patients with advanced non-small-cell lung cancer.Methods A total of 76 patients aged 60 years or over diagnosed with non-small-cell lung cancer(NSCLC) with EGFR-sensitive mutations and under Icotinib treatment were enrolled in this study.FBW7 and MCL-1 mRNA expression levels in peripheral blood were detected by real-time quantitative PCR(RT-QPCR).The correlation of FBW7 and MCL-1 expression levels with clinical and histological parameters,overall survival (OS),and progression-free-survival (PFS) was analyzed.Results The FBW7 expression level and the MCL-1 expression level were negative correlated(r =-0.37,P ＜0.001).High FBW7 expression levels and low MCL-1 expression levels in peripheral blood were associated with improved therapeutic efficacy of Icotinib (P＜0.001) and extended OS and PFS.Cox regression analysis showed that the expression levels of FBW7 and MCL-1 in peripheral blood were independent influencing factors for OS and PFS.Conclusions Patients with high FBW7 expression levels and low MCl-1 expression levels are more likely to benefit from Icotinib treatment.Expression levels for either factor can be used as a predictive indicator for the effectiveness of Icotinib and provide guidance for its clinical use.