Objective To investigate whether p38 mitogen activated protein kinase (p38MAPK) inhibitor can reduce acute lung injury (ALI) caused by lipopolysaccharide (LPS) by regulating Th17/Treg balance. Methods Balb/c mice were randomly divided into a control group, an ALI group and an intervention group. The mice in the control group were injected with phosphate-buffered saline, the mice in the ALI group were intraperitoneally injected with 40 mg/kg LPS, and the mice in the intervention group were injected with SB203580 (0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg) intraperitoneally 1 h prior to the intraperitoneal injection of LPS. All mice were killed on 12 h later respectively. Hematoxylin-eosinstin staining was used to observe the pathological changes of lung tissue, and cell classification, counting, and total protein levels in bronchoalveolar lavage fluid (BALF) were detected. Transcript expression of forkhead box p3 (Foxp3) and retinoic acid receptor-related orphan receptor-γt (RORγt) was detected by real-time polymerase chain reaction. Interleukin (IL)-6, IL-10, IL-17, IL-23 and transforming growth factor-β (TGF-β) in lung tissue and IL-6, tumor necrosis factor-α (TNF-α) in serum were measured by enzyme-linked immunosorbent assay. The Th17 and Treg subset distribution in spleen was determined by flow cytometry. Results Histopathological examination showed that LPS induced inflammatory cell infiltration in lung tissue, increased cell count and protein levels in BALF (P<0.05), and increased proportion of neutrophils and monocytes in the ALI mice. SB203580 significantly attenuated tissue injury of the lungs in LPS-induced ALI mice. Serum levels of IL-6 and TNF-α in the ALI group were significantly higher than those in the control group, and inflammatory cytokines were decreased after SB203580 intervention. Compared with the ALI group, the production of inflammatory cytokines associate with Th17, including IL-17, IL-23, RORγt was inhibited, and the production of cytokines associate with Treg, such as IL-10 and Foxp3 in lung tissue was increased in the intervention group in a concentration-dependent manner with SB203580. After SB203580 intervention, Th17/Treg ratio was significantly decreased compared with the LPS group (P<0.05). Conclusion p38MAPK inhibitor can reduce LPS-induced ALI by regulating the imbalance of Treg cells and Th17 cells.
ObjectiveTo study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function.MethodsBone marrow-derived macrophages (BMMs) were extracted from the marrow of femur and tibia of 4-week-old male C57BL/6 mice. Flow cytometry was used to detect BMMs. The effect of different concentrations of LPS (0, 100, 200, 500, 1 000, 2 000 ng/mL) on BMMs activity was examined by cell counting kit 8 (CCK-8) activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony-stimulating factor (M-CSF) group, M-CSF+receptor activator of nuclear factor κB ligand (RANKL) group, M-CSF+RANKL+50 ng/mL LPS group, M-CSF+RANKL+100 ng/mL LPS group. After the completion of culture, tartrate resistant acid phosphatase (TRAP) staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group (M-CSF+RANKL) and the LPS group (M-CSF+RANKL+100 ng/mL LPS); and the control group (M-CSF+RANKL), 50 ng/mL LPS group (M-CSF+RANKL+50 ng/mL LPS), and 100 ng/mL LPS group (M-CSF+RANKL+100 ng/mL LPS). The expressions of Connexin43 mRNA and protein were detected by Western blot and real-time fluorescent quantitative PCR, respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group. Bone absorption test was used to detect the ratio of bone resorption area.ResultsThe flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/mL LPS could promote the proliferation of BMMs, showing significant differences when compared with the 0, 200, 500, 1 000, and 2 000 ng/mL LPS (P<0.05). TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF+RANKL group, the osteoclasts in M-CSF+RANKL+50 ng/mL LPS group and M-CSF+RANKL+100 ng/mL LPS group were larger with more nuclei, while the osteoclasts in M-CSF+RANKL+100 ng/mL LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant (P<0.05). Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group (P<0.05). Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 mRNA in control group, 50 ng/mL LPS group, and 100 ng/mL LPS group increased gradually, and the differences between groups were statistically significant (P<0.05). Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group, and the differences between groups were statistically significant (P<0.05).ConclusionLPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.
ObjectiveTo study the effects of hydroxysafflow yellow A (HSYA) in inhibiting inflammatory signal transduction in lungs of acute lung injury mice induced by lipopolysaccharide (LPS). MethodsEighty-four male Kunming mice were randomly divided into 7 groups, ie. a sham group, a LPS group, a LPS+3 mg/kg dexamethason (DXM) group, a LPS+6 mg/kg HSYA group, a LPS+15 mg/kg HSYA group, a LPS+37.5 mg/kg HSYA group, and a saline+37.5 mg/kg HSYA group (n=12 in each group). The mice were intraperitoneally pretreated with normal saline or DXM or HSYA 0.5 hour prior to intravenous adminstration of LPS. TNF-α, IL-1β and IL-6 levels in mice serum were measured by ELISA and the mRNA and protein levels of TLR4 in mice lungs were assessed by RT-qPCR and Western blot, respectively. ResultsAfter being treated with HSYA in doses of 6 mg/kg, 15 mg/kg, and 37.5 mg/kg, the increased expression levels of TLR4 mRNA and protein induced by LPS were significantly inhibited, as well as the increased expression levels of TNF-α, IL-1β and IL-6. The inhibitoty effect enhanced with the doses of HSYA. DXM could inhibit more significantly the increased expression levels of all the indexes. ConclusionHSYA can inhibit inflammatory signal transduction in acute lung injury mice induced by LPS in a dose-dependent manner, but is less effective than DXM.
ObjectiveTo investigate the protective effect and mechanism of curcumin on lipopolysaccharide (LPS)-induced acute lung injury.MethodsTotally 24 SD rats were randomly divided into a control group, a LPS group and a LPS+curcumin group (n=8 in each group). The degree of lung injury (oxygen partial pressure, wet/dry ratio, pathological scores) and inflammatory levels [tumor necrosis factor (TNF)-α, interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, Toll-like receptor 4 (TLR4), mobility group box 1 protein (HMGB1) expression] of the lung were detected in different groups.ResultsOxygen partial pressure was significantly lower in the LPS group than that in the control group (P<0.05), while wet/dry ratio, pathological scores and expression levels of TNF-α, IL-6, MCP-1, TLR4 and HMGB1 were significantly higher in the LPS group than those in the control group (P<0.05). Compared with the LPS group, curcumin significantly reduced wet/dry ratio, pathological scores and expression levels of TNF-α, IL-6, MCP-1, TLR4 and HMGB1 in the LPS+curcumin group (P<0.05), while it significantly improved oxygen partial pressure (P<0.05).ConclusionCurcumin might protect LPS-induced acute lung injury through inhibition of TLR4-HMGB1-inflammation pathway.
Objective To investigate the effects of wedelolactone (WEL) on lipopolysaccharide (LPS)-induced pyroptosis of alveolar epithelial cells and AMP-activated protein kinase/nucleotide binding oligomeric domain like receptor 3 (NLRP3)/cysteinyl aspartate specific proteinase-1 (Caspase-1) signaling pathway. Methods Human lung epithelial cells BEAS-2B were treated with 5 - 200 μmol/L wedelolactone, and cell activity was detected using MTT assay. The alveolar epithelial cells were divided into control group, lipopolysaccharide group (LPS group), 10 μmol/L wedelolactone group (WEL-L group), 20 μmol/L wedelolactone group (WEL-M group), 40 μmol/L wedelolactone group (WEL-H group), 40 μmol/L wedelolactone+10 μmol/L AMPK inhibitor Compound C group (WEL-H+Compound C group), and 20 μmol/L Caspase-1 inhibitor Z-YVAD-FMK group (Z-YVAD-FMK group). Transmission electron microscopy was applied to observe the microstructure of cells. ELISA was applied to detect levels of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-8 (IL-8). Immunofluorescence was applied to detect Caspase-1 and gasdermin family proteins (DGSDMD). Western blot was applied to detect protein expression levels of AMPK, NLRP3, and Caspase-1. Results Wedelolactone concentrations of 10, 20 and 40 μmol/L were selected for follow-up experiments. Compared with Control group, LPS group showed decreased cell activity, severe damage, cell contraction, mitochondrial ridge breakage and decreased number, increased levels of TNF-α, IL-1β, IL-8 and GSDMD, NLRP3, Caspase-1 expression, and decreased p-AMPK/AMPK expression (P<0.05). Wedelolactone treatment could significantly improve LPS-induced pyrosis of alveolar epithelial cells (P<0.05). Compound C could partially reverse the effect of wedelactone on LPS-induced pyrodeath of alveolar epithelial cells (P<0.05). Z-YVAD-FMK treatment also significantly improved LPS-induced pyroptosis of alveolar epithelial cells (P<0.05). Conclusion Wedelolactone can inhibit LPS-induced pyroptosis of pulmonary alveolar epithelial cells by inhibiting AMPK/NLRP3/Caspase-1 signaling pathway.
ObjectiveTo investigate the effects of melatonin (MT) on bone mass and serum inflammatory factors in rats received ovariectomy (OVX) and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) stimulated by lipopolysaccharide. Methods Fifteen 12-week-old Sprague Dawley (SD) rats were randomly divided into 3 groups. The rats in Sham group only received bilateral lateral abdominal incision and suture, the rats in OVX group received bilateral OVX, and the rats in OVX+MT group received 100 mg/(kg·d) MT oral intervention after bilateral OVX. After 8 weeks, the levels of serum inflammatory factors [interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)] were detected using ELISA assay. Besides, the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure, and quantitatively measured bone volume fraction, trabecular thickness, and trabecular number. The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged. The 3rd-5th passage BMSCs were cultured with different concentrations of MT (0, 1, 10, 100, 1 000 µmol/L), and the cell viability was then detected using cell counting kit 8 (CCK-8) to select the optimal concentration of MT for subsequent experiments. Cells were devided into osteogenic induction group (group A) and osteogenic induction+1/5/10 μg/mL lipopolysaccharide group (group B-D). The levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in cell culture medium were detected using ELISA assay after corresponding intervention. According to the results of CCK-8 method and ELISA detection, the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction, defining as group E, and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay. After that, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively in groups A, D, and E, and the expression levels of osteogenic related genes [collagen type Ⅰ alpha 1 chain (Col1a1) and RUNX family transcription factor 2 (Runx2)] were also detected by real time fluorescence quantitative PCR (RT-qPCR). ResultsELISA and Micro-CT assays showed that compared with Sham group, the bone mass of the rats in the OVX group significantly decreased, and the expression levels of serum inflammatory factors (IL-1β, IL-6, and TNF-α) in OVX group significantly increased (P<0.05). Significantly, the above indicators in OVX+MT group were all improved (P<0.05). Rat BMSCs were successfully extracted, and CCK-8 assay showed that 100 µmol/L was the maximum concentration of MT that did not cause a decrease in cell viability, and it was used in subsequent experiments. ELISA assays showed that compared with group A, the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation (P<0.05), and in a concentration-dependent manner. Moreover, the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C (P<0.05). After MT intervention, the expression levels of inflammatory factors in group E were significantly lower than those in group D (P<0.05). ALP staining, alizarin red staining, and RT-qPCR assays showed that compared with group A, the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased, while the above indicators in group E significantly improved after MT intervention (P<0.05). ConclusionMT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats; MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.
Objective To investigate whether interleukin-1 receptor associated kinase (IRAK) participates the airway mucus hypersecretion induced by lipopolysaccharide (LPS). Methods The expression of Toll-like receptor (TLR)-4 or IRAK was down regulated by the transfection of specific small interfering RNA (siRNA). The transcription level of mucin 5AC (MUC5AC) mRNA as well as the secretion level of MUC5AC protein were judged by reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The activity of signaling molecules involved in TLR-4 associated pathway, such as the phosphorylated IRAK, nuclear translocation of NF-κB p65, was analyzed by Western blot. Results The level of intracellular phosphorylated IRAK was increased by stimulation of LPS in BEAS-2B cells. However, down-regulation of TLR-4 by siRNA could partially attenuate the additional phosphorylation of IRAK induced by LPS (P<0.05). LPS elicited a nuclear translocation of NF-κB p65 in BEAS-2B cells, which could be partially blocked by the down-regulation of TLR-4 or IRAK. With RT-PCR, an increased expression level of MUC5AC mRNA was discovered in wild-type BEAS-2B cells (0.82±0.09) than TLR-4 defect cells (0.36±0.05), IRAK defect cells (0.48±0.05) and IRAK inhibitor pretreated cells (0.57±0.07) (all P<0.05). Meanwhile, according to ELISA, an increased secretion level of MUC5AC protein was recorded in wild-type BEAS-2B cells [(0.76±0.09) μg/L] than TLR-4 defect cells [(0.33±0.04) μg/L], IRAK defect cells [(0.42±0.05) μg/L] and IRAK inhibitor pretreated cells [(0.51±0.06) μg/L] (all P<0.05). Conclusion As an crucial regulator of TLR dependent signaling pathway, IRAK may participate the airway mucus over-synthesis induced by LPS.
Objective To explore whether microRNA-203 (miR-203) targets and regulates the Toll-like receptor 4 (TLR4)/nuclear transcription factor kappa B (NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) to protect alveolar epithelial cells from lipopolysaccharide (LPS)-induced apoptosis and inflammation injury. Methods The alveolar epithelial A549 cells were used as the research objects and divided into: Control group (normal culture), LPS group (LPS treatment), LPS+miR-NC mimics group (LPS treatment after transfection of miR-NC mimics), LPS+ miR-203 mimics group (LPS treatment after transfection of miR-203 mimics), LPS+miR-203 mimics+pcDNA group (LPS treatment after transfection of miR-203 mimics and pcDNA), LPS+miR-203 mimics+pcDNA-TLR4 group (LPS treatment after transfection of miR-203 mimics and pcDNA-TLR4). Dual luciferase reporter gene was used to detect the targeting relationship between miR-203 and TLR4; Real-time quantitative reverse transcription-polymerase chain reaction was used to detect the relative expression levels of miR-203 and TLR4 mRNA; enzyme-linked immunosorbent assay was used to measure the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6; flow cytometry was used to detect the apoptosis rate of A549 cells; Western blot was used to detect the expression of B-cell lymphoma/leukemia-2 gene (Bcl-2) and Bcl-2 associated X protein (Bax), TLR4, NF-κB and NLRP3 proteins in A549 cells. Results There was a targeted regulation relationship between miR-203 and TLR4. Compared with the Control group, the expression of miR-203, TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS group increased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant increased, the apoptosis rate increased, the level of Bcl-2 protein in cells decreased (P<0.05). Compared with the LPS+miR-NC mimics group, the expression of TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS+miR-203 mimics group decreased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant decreased, the apoptosis rate decreased, the expression level of miR-203 and the level of Bcl-2 protein in cells increased (P<0.05). Compared with the LPS+miR-203 mimics+pcDNA group, the expression of miR-203, TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS+miR-203 mimics+pcDNA-TLR4 group increased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant increased, the apoptosis rate increased, the expression level of miR-203 and the level of Bcl-2 protein in cells decreased (P<0.05). Conclusion MiR-203 can target TLR4/NF-κB/NLRP3 to protect alveolar epithelial cells from apoptosis and inflammation induced by LPS.
ObjectiveTo investigate the effect of curcumin on the expression regulation of endogenousβ-glucoronidase (β-GD) induced by lipopolysaccharide (LPS).Methods① Human normal intrahepatic biliary epithelial cell line (HiBEpiC) cells in the logarithmic growth phase were divided into blank control group (0 h group) and 7 different stimulation time groups. The cell density was adjusted to 1×104/mL, and the cells were stimulated with 100 mg/mL LPS for 1, 3, 6, 18, and 24 hours respectively, including another two groups where the cells were cultured with LPS-free medium for 18 and 24 hours after LPS stimulation for 24 h. ② HiBEpiC cells in the logarithmic growth phase were divided into blank control group, LPS+low, medium, and high concentration curcumin group. The cell density was adjusted to 1×104/mL. In the blank control group, cells were not stimulated with any reagent; in the LPS group, cells were stimulated with 100 mg/mL LPS, in the other three groups, the cells were stimulated with 100 mg/mL LPS and simultaneously 20, 40, and 80 μmol/L curcumin, respectively, for 24 hours. The expressions of c-myc and endogenous β-GD were detected by Western blot method.Results① The expressions of endogenous β-GD and c-myc in HiBEpiC cells gradually increased with the prolongation of treatment time by LPS, and the expression levels of β-GD and c-myc at each time point group were significantly different from those in the 0 h group (P<0.05). ② There were significant difference between any two groups of the blank control group, LPS group, LPS+low concentration of curcumin group, LPS+medium concentration of curcumin group, and LPS+high concentration of curcumin group (P<0.05).ConclusionCurcumin is able to inhibit the increased expression of endogenous β-GD induced by LPS, possibly via inhibiting expression of c-myc.
Objective To investigate the ability of gene-loaded lipopolysaccharide-amine nanopolymersomes (LNPs) in inducing osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by in vitro gene transfection, where LNPs were used as a non-viral cationic carrier, and their properties were optimized during synthesis. Methods LNPs were synthesized by a graft-copolymerization method, and the effects of different pH environments during synthesis on physicochemical properties of LNPs and LNPs/plasmid of bone morphogenetic protein 2-green fluorescent protein (pBMP-2-GFP) complexes were explored. Then, optimized LNPs with maximum transfection efficiency and safe cytotoxicity in rat BMSCs were identified by cytotoxicity and transfection experiments in vitro. Thereafter, the optimized LNPs were used to mediate pBMP-2-GFP to transfect rat BMSCs, and the influences of LNPs/pBMP-2-GFP on osteogenic differentiation of BMSCs were evaluated by monitoring the cell morphology, concentration of BMP-2 protein, activity of alkaline phosphatase (ALP), and the formation of calcium nodules. Results The nitrogen content, particle size, and zeta potential of LNPs synthesized at pH 8.5 were lower than those of the other pH groups, with the lowest cytotoxicity (96.5%±1.4%) and the highest transfection efficiency (98.8%±0.1%). After transfection treatment, within the first 4 days, BMSCs treated by LNPs/pBMP-2-GFP expressed BMP-2 protein significantly higher than that treated by Lipofectamine2000 (Lipo)/pBMP-2-GFP, polyethylenimine 25K/pBMP-2-GFP, and the blank (non-treated). At 14 days after transfection, ALP activity in BMSCs treated by LNPs/pBMP-2-GFP was higher than that treated by Lipo/pBMP-2-GFP and the blank, comparable to that induced by osteogenic medium; with alizarin red staining, visible calcium nodules were found in BMSCs treated by LNPs/pBMP-2-GFP or osteogenic medium, but absent in BMSCs treated by Lipo/pBMP-2-GFP or the blank with apoptosis. At 21 days after transfection, transparent massive nodules were discovered in BMSCs treated by LNPs/pBMP-2-GFP, and BMSCs exhibited the morphologic features of osteoblasts. Conclusion LNPs synthesized at pH 8.5 has optimal transfection efficiency and cytotoxicity, they can efficiently mediate pBMP-2-GFP to transfect BMSCs, and successfully induce their directional osteogenic differentiation, whose inducing effect is comparable to the osteogenic medium. The results suggest that gene transfection mediated by LNPs may be a convenient and effective strategy in inducing directional differentiation of stem cells.