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BACKGROUND: Osteoporosis has a high incidence, leading to fracture and other complications. However, existing drugs have great side effects and are difficult to meet the clinical application. OBJECTIVE: To explore the effect and potential mechanism of fucoxanthin on osteoporosis induced by glucocorticoid. METHODS: Primary rat osteoblasts were inoculated in 6-well plates. When the cell fusion reached 80%, the cells were divided into four groups: the control group was cultured alone for 24 hours, the glucocorticoid group was intervened with dexamethasone for 24 hours, the fucoxanthin group was intervened with fucoxanthin for 24 hours, and the glucocorticoid + fucoxanthin group was intervened with dexamethasone and fucoxanthin at the same time for 24 hours. After intervention, cell proliferation, apoptosis, intracellular reactive oxygen species level, and protein expression of apoptosis-related proteins, bone formationrelated proteins, and nuclear factor erythroid-2-related factor 2 were detected. RESULTS AND CONCLUSION: Cell counting kit-8 results showed that the cell viability was decreased in the glucocorticoid group compared with the control group (P < 0.05) but increased in the glucocorticoid+fucoxanthin group compared with the glucocorticoid group (P < 0.05). JC-1 mitochondrial membrane potential staining and flow cytometry assay showed that the percentage of apoptosis increased in the glucocorticoid group compared with the control group (P < 0.05) but decreased in the glucocorticoid+fucoxanthin group compared with the glucocorticoid group (P < 0.05). Western blot assay showed that compared with the control group, the protein expression of BAX and cleaved poly (ADP-ribose) polymerase was elevated in the glucocorticoid group (P < 0.05), and the protein expression of BCL2, type I collagen α1 peptide chain, alkaline phosphatase, osteocalcin, and RUNX2 was decreased in the glucocorticoid group (P < 0.05). Compared with the glucocorticoid group, the protein expression of BAX and cleaved poly (ADP-ribose) polymerase was decreased (P < 0.05), and the protein expression of BCL2, type I collagen α1 peptide chain, alkaline phosphatase, osteocalcin, and RUNX2 was elevated (P < 0.05) in the glucocorticoid+fucoxanthin group. Fluorescent probe assay showed an increase in reactive oxygen species level in the glucocorticoid group compared with the control group (P < 0.05) and a decrease in reactive oxygen species level in the glucocorticoid+fucoxanthin group compared with the glucocorticoid group (P < 0.05). Immunofluorescence staining and western blot assay showed that the protein expression of nuclear factor erythroid-2-related factor 2 in the glucocorticoid group was decreased compared with that in the control group (P < 0.05); and the protein expression of nuclear factor erythroid-2-related factor 2 in the glucocorticoid+fucoxanthin group was elevated compared with that in the glucocorticoid group (P < 0.05). To conclude, fucoxanthin can improve glucocorticoid-induced osteoblast apoptosis and the expression of bone formation-related molecules by activating nuclear factor erythroid-2-related factor 2. [ABSTRACT FROM AUTHOR]

[Objectives]The paper aimed to investigate the specific mechanism through which isoflurane attenuated inflammation induced by microglia hyperactivation. [Methods]The microglia cells were treated by lipopolysaccharide(LPS)inducing inflammatory response in vitro, and the prepared emulsified isoflurane was used to treat to the cells. The effects of isoflurane and pathway protein inhibitors on the expression of pro-inflammatory mediators(pro-inflammatory factors, pro-inflammatory enzymes)and pathway proteins were detected by fluorescence quantitative PCR and Western blot, and then the effects of different inhibitors on protein expression and the signaling pathway of isoflurane action were determined. At the same time, the effects of different treatments on the translocation of nuclear factor κB(NF-κB)in microglia cells were observed. [Results]Compared with the control group, LPS significantly increased the expressions of interleukin(IL)-1β, IL-6, nitric oxide synthase(iNOS), cyclooxygenase-2(COXⅡ)and other pro-inflammatory factors, and up-regulated the expressions of target pathway protein and the translocation of NF-κB from cytoplasm to nucleus. Isoflurane reduced the expression of pro-inflammatory media and inhibited NF-κB nuclear translocation through CaMKⅡ/ERK/NF-κB signaling pathway. [Conclusions]Isoflurane reduced the release of proinflammatory factors and alleviated the inflammatory response of microglia cells through Ca2+ signaling pathway. [ABSTRACT FROM AUTHOR]

ObjectiveTo investigate the molecular mechanism of how lactate induces high mobility group box 1 (HMGB1) release. MethodsGastric cancer HGC-27 cells were divided into the control group and the lactate group (The cells were treated with lactate for 6 h). The level of HMGB1 in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA), the localization of HMGB1 was detected using laser confocal microscopy, and the nuclear translocation of HMGB1 was detected using the nucleoplasmic separation assay. The phosphorylation and acetylation levels of HMGB1 were determined by co-immunoprecipitation, and Western blot was used to measure the phosphorylation of Akt and protein kinase C (PKC). HGC-27 cells were first treated with lactate and LY294002, the inhibitor of Akt, and then the phosphorylation of HMGB1 and Akt was analyzed by co-immunoprecipitation and Western blot, respectively. The localization of HMGB1 in cells was detected by laser confocal microscopy. EdU and Transwell assays were used to detect the proliferation and migration abilities of HGC-27 cells, respectively. HGC-27 cells were then injected into the BALB/C null mice for subcutaneous tumor implantation. Mice in the lactate group were intraperitoneally injected with lactate (0.2 g/kg/2 d), while those in the control group were intraperitoneally injected with an equal amount of PBS for 20 consecutive days. ELISA was used to detect the HMGB1 levels in the blood samples taken from the medial canthus vein of the mice, while co-immunoprecipitation and Western blot were used to detect the phosphorylation of HMGB1 and Akt in tumor tissue proteins, respectively. ResultsThe release levels of HMGB1 in the lactate group were (2 995.00±660.91) pg/ml and (696.33±22.03) pg/ml, after lactate treatment for 6 h and 12 h, respectively, both higher than those in the control group (485.00±105.83) pg/ml (P<0.001 and P=0.028, respectively). After lactate treatment for 6 h, the relative expression of HMGB1 protein in the cytoplasm of HGC-27 cells was 1.13±0.09, higher than that of the control group (0.83±0.07, P=0.001), while the relative expression of HMGB1 in the nucleus was 0.79±0.06, lower than that of the control group (1.07±0.06, P=0.007). The phosphorylation level of HMGB1 reached 1.41±0.09, which was higher than that of the control group (0.97±0.10, P=0.031). The phosphorylation level of Akt was 11.16±0.06, higher than that of the control group (0.91±0.022, P=0.002). The phosphorylation level and nuclear translocation of HMGB1 induced by lactate decreased obviously after Akt inhibition; the proliferation and migration abilities induced by lactate were also obviously inhibited after Akt inhibition. In vivo, the HMGB1 level in the peripheral blood was (1 280.70±389.66) pg/ml in the lactate group, which was obviously higher than that in the control group (595.11±44.75) pg/ml (P=0.008), and the phosphorylation levels of HMGB1 and Akt in tumor tissues in the lactate group were obviously enhanced compared with the control group. ConclusionLactate induces HMGB1 release through enhancing HMGB1 phosphorylation via the Akt signaling pathway. [ABSTRACT FROM AUTHOR]

BackgroundParaquat (PQ), one of the environmental poisons associated with sporadic Parkinson's disease (PD), can cause abnormal aggregation of alpha-synuclein (α-syn), but the research on its conformational changes and subcellular localization is limited.ObjectiveTo investigate the effect of PQ on α-syn conformation and subcellular localization in dopaminergic neurons.MethodsForty-eight SPF C57BL/6 male mice were selected and randomly divided into a control group and a model group. The model group was intraperitoneally injected with PQ (15 mg·kg−1), and the control group was intraperitoneally injected with 0.9% normal saline, twice a week for eight weeks to construct a PD-like mouse model. The changes of neurobehavior (by open field test and pole climbing test) were observed to evaluate motor ability of mice. Immunohistochemical staining (IHC) was used to detect the expression levels of tyrosine hydroxylase (TH) and α-syn in the midbrain. Western blotting (WB) was used to measure the protein expression levels of TH and α-syn in midbrain. Human neuroblastoma SH-SY5Y cells were used as dopaminergic neuron in vitro models. After the cells were treated with PQ (100 μmol·L−1) for 0, 12, 24, 36 and 48 h, the expressions of α-syn in whole cell, cytoplasm, and nucleus were detected by WB; the expression level of extracellular α-syn was detected by enzyme-linked immunosorbent assay (ELISA); the change of α-syn location was observed by immunofluorescence assay (IFA).ResultsThe neurobehavioral tests' results showed that compared with the control group, the residence time in peripheral area of mice in the PQ model group increased with the increase of exposure time (P

M1 macrophages are classical activated macrophages, which can secrete a variety of pro-inflammatory factors and chemokines to promote the development of inflammation. Our previous studies have demonstrated that 630 nm light-emitting diode (LED) irradiation inhibits the release of inflammatory factors in macrophages. However, the effect of 630 nm LED on macrophage polarization and its molecular mechanism remains unclear. In this study, we evaluated the effect of 630 nm LED irradiation on the polarization of THP-1 derived M1 macrophages and explored the mechanism of its effect on the polarization of M1 macrophages. Human mononuclear cell line THP-1 was cultured in vitro and induced into macrophages by phorbol myristate acetate (PMA). Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) treated cells were polarized into M1 macrophages, which were irradiated by LED with a wavelength of 630 nm and a power density of 40.02 mW/cm2 . CCK-8 colorimetry, MTT assay and lactate dehydrogenase (LDH) activity detection kit were used to detect cell viability. Real-time quantitative PCR was used to detect mRNA expression levels of related factors in M1 macrophages. Western blot and immunofluorescence were used to detect the nuclear translocation of STAT1 in M1 macrophages. The results showed that the cell viability of M1 macrophages did not alter after 630 nm LED irradiation. LED irradiation significantly reduced the mRNA expression levels of the interferon regulatory factor 5 (IRF5) and C-X-C motif chemokine ligand (CXCL) 9, 10 and 11 and block the translocation of transcription factor STAT1. Taken together, 630 nm LED inhibits the polarization of M1 macrophages by blocked STAT1 nuclear translocation. This study may provide new therapeutic ideas for the treatment of diseases related to macrophage polarization. M1 macrophages are classical activated macrophages, which can secrete a variety of pro-inflammatory factors and chemokines to promote the development of inflammation. Our previous studies have demonstrated that 630 nm light-emitting diode (LED) irradiation inhibits the release of inflammatory factors in macrophages. However, the effect of 630 nm LED on macrophage polarization and its molecular mechanism remains unclear. In this study, we evaluated the effect of 630 nm LED irradiation on the polarization of THP-1 derived M1 macrophages and explored the mechanism of its effect on the polarization of M1 macrophages. Human mononuclear cell line THP-1 was cultured in vitro and induced into macrophages by phorbol myristate acetate (PMA). Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) treated cells were polarized into M1 macrophages, which were irradiated by LED with a wavelength of 630 nm and a power density of 40.02 mW/cm2 . CCK-8 colorimetry, MTT assay and lactate dehydrogenase (LDH) activity detection kit were used to detect cell viability. Real-time quantitative PCR was used to detect mRNA expression levels of related factors in M1 macrophages. Western blot and immunofluorescence were used to detect the nuclear translocation of STAT1 in M1 macrophages. The results showed that the cell viability of M1 macrophages did not alter after 630 nm LED irradiation. LED irradiation significantly reduced the mRNA expression levels of the interferon regulatory factor 5 (IRF5) and C-X-C motif chemokine ligand (CXCL) 9, 10 and 11 and block the translocation of transcription factor STAT1. Taken together, 630 nm LED inhibits the polarization of M1 macrophages by blocked STAT1 nuclear translocation. This study may provide new therapeutic ideas for the treatment of diseases related to macrophage polarization. [ABSTRACT FROM AUTHOR]

Objective: To investigate the molecular mechanism of how lactate induces high mobility group box 1 (HMGB1) release. Methods: Gastric cancer HGC-27 cells were divided into the control group and the lactate group (The cells were treated with lactate for 6 h). The level of HMGB1 in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA), the localization of HMGB1 was detected using laser confocal microscopy, and the nuclear translocation of HMGB1 was detected using the nucleoplasmic separation assay. The phosphorylation and acetylation levels of HMGB1 were determined by co-immunoprecipitation, and Western blot was used to measure the phosphorylation of Akt and protein kinase C (PKC). HGC-27 cells were first treated with lactate and LY294002, the inhibitor of Akt, and then the phosphorylation of HMGB1 and Akt was analyzed by co-immunoprecipitation and Western blot, respectively. The localization of HMGB1 in cells was detected by laser confocal microscopy. EdU and Transwell assays were used to detect the proliferation and migration abilities of HGC-27 cells, respectively. HGC-27 cells were then injected into the BALB/C null mice for subcutaneous tumor implantation. Mice in the lactate group were intraperitoneally injected with lactate (0.2 g/kg/2 d), while those in the control group were intraperitoneally injected with an equal amount of PBS for 20 consecutive days. ELISA was used to detect the HMGB1 levels in the blood samples taken from the medial canthus vein of the mice, while co-immunoprecipitation and Western blot were used to detect the phosphorylation of HMGB1 and Akt in tumor tissue proteins, respectively. Results: The release levels of HMGB1 in the lactate group were (2 995.00±660.91) pg/ml and (696.33±22.03) pg/ml, after lactate treatment for 6 h and 12 h, respectively, both higher than those in the control group (485.00±105.83) pg/ml ( P <0.001 and P =0.028, respectively). After lactate treatment for 6 h, the relative expression of HMGB1 protein in the cytoplasm of HGC-27 cells was 1.13±0.09, higher than that of the control group (0.83±0.07, P =0.001), while the relative expression of HMGB1 in the nucleus was 0.79±0.06, lower than that of the control group (1.07±0.06, P =0.007). The phosphorylation level of HMGB1 reached 1.41±0.09, which was higher than that of the control group (0.97±0.10, P =0.031). The phosphorylation level of Akt was 11.16±0.06, higher than that of the control group (0.91±0.022, P =0.002). The phosphorylation level and nuclear translocation of HMGB1 induced by lactate decreased obviously after Akt inhibition; the proliferation and migration abilities induced by lactate were also obviously inhibited after Akt inhibition. In vivo, the HMGB1 level in the peripheral blood was (1 280.70±389.66) pg/ml in the lactate group, which was obviously higher than that in the control group (595.11±44.75) pg/ml ( P =0.008), and the phosphorylation levels of HMGB1 and Akt in tumor tissues in the lactate group were obviously enhanced compared with the control group. Conclusion: Lactate induces HMGB1 release through enhancing HMGB1 phosphorylation via the Akt signaling pathway.

Objective To explore the impacts of over-expression of microRNA-7 (miRNA-7) on the sensitivity of cisplatin in esophageal carcinoma cell line TE-1, and the possible mechanism thereof. Methods Lipofectmin 2000 method was used to transient transfect with miRNA-7 mimic into esophageal cancer cell line TE-1, which was taken as transfection group, mimic negative control was taken as transfection conrtol group. The expressions of miRNA-7 and epidermal growth factor receptor (EGFR) mRNA were detected by RT-PCR in the above two groups and normal control group. The total EGFR and EGFR in cytoplasmic and nucleus were detected with Western blot assay in transfection group and transfection control group. CCK-8 was used to detect IC50 of cisplatin in transfection group and transfection control group. The expression of EGFR was observed with immunofluorescence confocal microscope in two groups. Results The miRNA-7 expression was significantly increased in transfection group than that of transfection conrtol group and control group. The expression of EGFR mRNA was significantly reduced in transfection group (P＜0.001). The total EGFR was significantly decreased in transfection group than that of transfection conrtol group. The level of nuclear EGFR was significantly increased (P＜0.01)，and cytoplasm EGFR expression was significantly decreased in transfection group than that of transfection control group (P＜0.05). CCK-8 results showed that after the over expression of miRNA-7 in TE-1, the IC50 of cisplatin (48 h) increased in transfection group than that of control group (P＜0.01). Immunofluorescence results showed that EGFG in nuclear was higher in transfection group than that of transfection control group but its expressions reduced in cell membrane and cytoplasm. Conclusion The over-expressed miRNA-7 in esophageal cancer cells TE-1 can reduce cisplatin sensitivity by the increased EGFR in nuclear translocation. [ABSTRACT FROM AUTHOR]

Objective: The effect of total flavonoids of litchi (TFL) on nuclear translocation of nuclear factor-kappa B (NF- kappa B) in rat hepatic stellate cell line (HSC-T6) induced by transforming growth factor - beta 1 (TGF- beta 1) in vitro was studied to explore the mechanism of action of anti-hepatic fibrosis drugs. Methods: HSC-T6 was cultured in vitro, induced by TGFβ1 for 24 h, and then treated with TFL at 125, 250 and 500 μg/ml for 48 h. The effect of TFL on NF-κB nuclear translocation in HSC-T6 was observed by confocal laser microscopy. The effects of TFL on the expression of TLR4, p-IκB ɑ, p-NF-κB p65, NF-κB and Collagen I protein were detected by western blot. The expressions of TLR4 and p-NF-κB p65 were detected by immunofluorescence. Data were presented as mean±SEM. Homogeneity test of variance was performed and then followed by one-way analysis of variance (ANOVA). The multiple comparisons between groups were performed by LSD test. P < 0.05 was considered statistically significant. Results: Confocal laser scanning microscopy showed TFL inhibited the nuclear translocation of NF-κB in activated HSC-T6 in a concentration-dependent manner and TFL down regulated the protein expression levels of TLR4, p-IκB ɑ, p-NF-κB p65, NF-κB and collagen I protein in HSC-T6 in a concentration-dependent manner. Conclusion: The mechanism of TFL against hepatic fibrosis may be related to the inhibition of nuclear translocation of NF-κb in the activated HSC-T6 and the expression of TLR4, P-iκbɑ, P-nf-κb p65, NF-κb and collagen I protein in HSC-T6.