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Background The mechanisms of silicon dioxide (SiO2)-induced inflammation and cell injury in pulmonary macrophages are not fully characterized. Objective To investigate the potential roles of inhibition of toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling in inflammation and macrophage polarization in mouse Raw264.7 cells in response to SiO2 stimulation. Methods Sixteen 6- to 8-week-old C57BL/6 mice, half male and half female, were intratracheally instilled with 50 µL of SiO2 (50 mg·mL−1 in saline) or normal saline via oropharyngeal route, and the lungs of mice were harvested at 14 d and 28 d post the first challenge of SiO2. HE staining of mouse lung was used for histopathological analysis. The expressions of TLR4 signaling-related proteins were detected by Western blotting (WB) and immunofluorescent (IF) assay, including TLR4, myeloid differentiation factor 88 (Myd88), and TNF receptor associated factor 6 (TRAF6). Raw264.7 cells were stimulated with SiO2 (100 μg·cm2) for 12 h in absence or presence of TLR4 inhibitor M62812 for 13 h before the culture supernatants and cell lysates were harvested for analysis. The expressions of key components of TLR4 signaling cascade including TLR4, Myd88, and phosphorylated nuclear factor-kappa B P65 (P-NF-κB P65), P-1NF-kappa-B inhibitor α (P-1κbα), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), M1 phenotype markers inducible nitric oxide synthase (iNOS) and cluster of differentiation 86 (CD86), as well as M2 phenotype arginase-1 (Arg-1) were accessed by WB and IF. The expressions of inflammation factors IL-6 and TNF-α in supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Results After SiO2 intratracheal instillation for 14 d, the HE staining results showed obvious fibrotic nodules in the lung tissues of mice. The results of WB analysis revealed more abundant TLR4, Myd88, and TRAF6 in the silicosis mouse lung samples than in the controls. The results of IF assay showed an increased abundance of TLR4 and Myd88 proteins in the lung samples of silicosis mice at 14 d post the silica challenge, compared to the controls, indicating TLR4 signaling activation. As seen in the in vitro experiment, significant upregulations after the exposure to 100 μg·cm2 SiO2 were observed in TLR4 and P-1κbα at 6, 12, and 24 h (P<0.05); Myd88 at 12 and 24 h (P <0.05); and P-NF-κB P65 at 12 h (P<0.05). The inhibitor significantly suppressed the expressions of TLR4, Myd88, TRAF6, P-NF-κB P65, TNF-α, and IL-6 in Raw264.7 cells. In addition, the SiO2-induced M1 phenotype marker iNOS was significantly suppressed, but the M2 phenotype marker Arg-1 was increased in the Raw264.7 cells. Conclusion The inhibition of TLR4/NF-κB signaling could result in a reduction of the inflammation response and the transition of M1 toward M2 phenotypes of macrophages in response to SiO2 challenge.
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Objective@#To investigate the drug resistance of kaempferol reversed adriamycin (ADM)-resistant K562/ADM cells in chronic myelogenous leukemia (CML) and its related mechanism.@*Methods@#Methyl thiazolyl tetrazolium (MTT) method was used to detect the toxicity of ADM on K562 and K562/ADM cells for 24 h. The half inhibitory concentration (IC50) of ADM and the drug resistance multiple for 24 h were calculated. MTT method was used to detect the toxicity of kaempferol on K562/ADM cells for 24 h. The 5% inhibitory concentration (IC5) and 10% inhibitory concentration (IC10) of kaempferol for 24 h were calculated to determine the concentration of kaempferol in the subsequent experiments. And the cells untreated by the kaempferol were selected as the control group. The cell inhibition after the treatment of ADM for 24 h of the blank control group and kaempferol intervention group was detected by using MTT method. And then the cell inhibition for 24 h and ADM IC50 for 24 h in the above groups were calculated. The ratio of IC50 in the blank control group and kaempferol group was the reversal drug resistance multiple of kaempferol. The fluorescence intensity of ADM in K562/ADM cells treated by kaempferol was detected by using flow cytometry. Western blotting was used to detect the expressions of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), phosphorylated p38 (p-p38), and total p38 (t-p38) protein in K562/ADM cells after the treatment of kaempferol, the specific inhibitor of p38-MAPK signaling pathway SB202190, and the combination of kaempferol and SB202190.@*Results@#After the treatment of ADM for 24 h, the IC50 value of K562 and K562/ADM cells was (0.9±0.6), (28.1 ±3.5) μg/ml, respectively. The drug resistance multiple of K562/ADM cells on the treatment of ADM for 24 h was 31.16 compared with the K562 cells. MTT method showed that kaempferol inhibited the proliferation of K562/ADM cells in a dose-dependent manner. According to the IC5 and IC10, 0.5 μmol/L and 1.0 μmol/L kaempferol were determined to do the subsequent experiments. After the combined interaction of kaempferol and ADM for 24 h, the ADM IC50 of K562/ADM cells in the blank control group, 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was (33.7±5.7), (21.4±0.6), (15.9±1.8) μg/ml, respectively (F = 30.85, P < 0.05), and there was a statistical difference of pairwise comparison (both P < 0.05). The reversal drug resistance multiple of K562/ADM cells for 24 h in 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was 1.58 and 2.12, respectively. Flow cytometry results showed that the mean fluorescence intensity (MFI) of ADM in the blank control group, 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was 138.4±8.9, 154.3±2.2, 165.7±4.8, respectively, and the difference was statistically significant (F = 161.48, P < 0.05). Compared with the blank control group, after treatment of K562/ADM cells with 0.5 μmol/L and 1.0 μmol/L kaempferol for 24 h, the relative expressions of P-gp, MRP1 and p-p38 protein were decreased in K562/ADM cells (all P < 0.05), but there was no statistical difference in the expression of t-p38 protein (P > 0.05); SB202190 could reduce the relative expressions of P-gp, MRP1 and p-p38 protein (all P < 0.05); after the treatment of SB202190 combined with different concentration of kaempferol, the relative expressions of P-gp, MRP1 and p-p38 protein in K562/ADM cells did not decrease (P > 0.05).@*Conclusions@#Kaempferol can decrease the relative expressions of P-gp and MRP1 in K562/ADM cells by inhibiting p38-MAPK pathway, so as to increase the concentrations of ADM and to reverse the drug resistance of K562/ADM cells.
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Objective:To investigate the drug resistance of kaempferol reversed adriamycin (ADM)-resistant K562/ADM cells in chronic myelogenous leukemia (CML) and its related mechanism.Methods:Methyl thiazolyl tetrazolium (MTT) method was used to detect the toxicity of ADM on K562 and K562/ADM cells for 24 h. The half inhibitory concentration ( IC50) of ADM and the drug resistance multiple for 24 h were calculated. MTT method was used to detect the toxicity of kaempferol on K562/ADM cells for 24 h. The 5% inhibitory concentration ( IC5) and 10% inhibitory concentration ( IC10) of kaempferol for 24 h were calculated to determine the concentration of kaempferol in the subsequent experiments. And the cells untreated by the kaempferol were selected as the control group. The cell inhibition after the treatment of ADM for 24 h of the blank control group and kaempferol intervention group was detected by using MTT method. And then the cell inhibition for 24 h and ADM IC50 for 24 h in the above groups were calculated. The ratio of IC50 in the blank control group and kaempferol group was the reversal drug resistance multiple of kaempferol. The fluorescence intensity of ADM in K562/ADM cells treated by kaempferol was detected by using flow cytometry. Western blotting was used to detect the expressions of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), phosphorylated p38 (p-p38), and total p38 (t-p38) protein in K562/ADM cells after the treatment of kaempferol, the specific inhibitor of p38-MAPK signaling pathway SB202190, and the combination of kaempferol and SB202190. Results:After the treatment of ADM for 24 h, the IC50 value of K562 and K562/ADM cells was (0.9±0.6), (28.1 ±3.5) μg/ml, respectively. The drug resistance multiple of K562/ADM cells on the treatment of ADM for 24 h was 31.16 compared with the K562 cells. MTT method showed that kaempferol inhibited the proliferation of K562/ADM cells in a dose-dependent manner. According to the IC5 and IC10, 0.5 μmol/L and 1.0 μmol/L kaempferol were determined to do the subsequent experiments. After the combined interaction of kaempferol and ADM for 24 h, the ADM IC50 of K562/ADM cells in the blank control group, 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was (33.7±5.7), (21.4±0.6), (15.9±1.8) μg/ml, respectively ( F = 30.85, P < 0.05), and there was a statistical difference of pairwise comparison (both P < 0.05). The reversal drug resistance multiple of K562/ADM cells for 24 h in 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was 1.58 and 2.12, respectively. Flow cytometry results showed that the mean fluorescence intensity (MFI) of ADM in the blank control group, 0.5 μmol/L kaempferol group and 1.0 μmol/L kaempferol group was 138.4±8.9, 154.3±2.2, 165.7±4.8, respectively, and the difference was statistically significant ( F = 161.48, P < 0.05). Compared with the blank control group, after treatment of K562/ADM cells with 0.5 μmol/L and 1.0 μmol/L kaempferol for 24 h, the relative expressions of P-gp, MRP1 and p-p38 protein were decreased in K562/ADM cells (all P < 0.05), but there was no statistical difference in the expression of t-p38 protein ( P > 0.05); SB202190 could reduce the relative expressions of P-gp, MRP1 and p-p38 protein (all P < 0.05); after the treatment of SB202190 combined with different concentration of kaempferol, the relative expressions of P-gp, MRP1 and p-p38 protein in K562/ADM cells did not decrease ( P > 0.05). Conclusions:Kaempferol can decrease the relative expressions of P-gp and MRP1 in K562/ADM cells by inhibiting p38-MAPK pathway, so as to increase the concentrations of ADM and to reverse the drug resistance of K562/ADM cells.
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Gastrointestinal microflora are the largest and the most complicated micro-ecosystem in hu-man′s body. It is parasitic on the human′s gastrointestinal tract,and help the host to finish many physiological and biochemical functions. The patients who are diagnosed with leukemia will have large change in their gastro-intestinal microflora. After allogeneic hematopoietic stem cell transplantation,patients can suffer from acute graft versus host disease,and studies have shown that gastrointestinal microflora play an important role in reducing the incidence of acute graft-versus-host disease. Maintaining the balance of gastrointestinal microflora and using it as the target for leukemia treatment can prolong the survival period of leukemia patients. This phenomenon mani-fests that the gastrointestinal microflora is closely associated with leukemia. It will provide new strategy to leuke-mia′s treatment by studying the correlation between gastrointestinal microflora and leukemia.
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Objective To explore the effect of proliferation and apoptosis of Solanine on acute T lymphocyte leukemia (T-ALL) Jurkat cells and its mechanism.Methods After treated with different concentrations of Solanine,the proliferation of Jurkat cells was detected by CCK-8 assay,and the effect of Solanine on apoptosis of Jurkat cells were detected by flow cytometry.The expression of Bcl-2 and Bax in Jurkat cells were detected by Western blot,and the expression levels of Bcl-2 mRNA and Bax mRNA were detected by real-time fluorescence quantitative polymerase chain reaction.Results CCK-8 assay showed that Solanine significantly inhibited the proliferation of Jurkat cells in a dose-and time-dependent manner.The results of flow cytometry showed that the apoptosis rates of Jurkat cells treated with Solanine for 24 h were (2.40-± 0.98) ％,(28.43-± 4.86) ％,(41.56-± 1.87) ％,respectively,in a dose-dependent manner.Western blot showed that Solanine could increase the expression of Bax and decrease the expression of Bcl-2 in Jurkat cells,and they all were dose-dependent.Conclusion Solanine can significantly inhibit the proliferation and induce apoptosis of Jurkat cells.The mechanism is related to the up-regulation of Bax expression and down-regulation of Bcl-2 expression.