c-kit+ cells are mainly derived from bone marrow and cardiac tissue.The cells include various subpopulations.Recent studies have shown that c-kit+ cells are a kind of ideal cells of transplantation therapy for myocardial infarction.However,there is a great debate on the differentiation efficiency of c-kit+ cells towards cardiomyocytes and endothelial cells.Therefore,it is necessary to evaluate the potential of c-kit+ cell differentiation and explore the effects of different developmental stages and microenvironments on differentiation of c-kit+ cells again.These studies could be significant for increasing efficiency of c-kit+ cell transplantation in repairing the infarcted myocardium.

Objective:To establish cell lines stably expressing Rab5a and its the inactive mutant Rab5aN133I,analyze the effect of Rab5a on the expression of cytokines in LPS-stimulated RAW264.7 cells .Methods:RAW264.7 cells were transfected with Rab5a and its inactive mutant vector Rab5a N133I separately,and then screened by G418.Rab5a stable expressing cell lines were identified by Real time-PCR.The growth of the stable cell lines was analyzed by MTT assay.After the stable cell lines were stimulated by LPS for different time periods,the expression of iNOS,TNF-αand IL-6 was detected.Results:Rab5a and Rab5aN133I transfection resulted in elevated Rab5a mRNA expression compared with the control cells ( P<0.05 ).Rab5a overexpression enhanced the proliferation of RAW264.7 cells.However,the proliferation of Rab5aN133I cells was significantly slower than the control cells ( P<0.05).Overexpression of Rab5a promoted LPS-induced production of iNOS,TNF-αand IL-6 in RAW264.7 cells (P<0.01). Conversely,overexpression of Rab5aN133I abolished the stimulating effects of Rab5a.Conclusion: Rab5a promoted LPS-induced expression of iNOS,TNF-αand IL-6 in RAW264.7 macrophages in a GTP-binding ability-dependent manner.

Objective To investigate effects of calycosin(CA)on cigarette smoke(CS)induced airway epithelial cell damage in mice and the sirtuin 3/superoxide dismutase 2(SIRT3/SOD2)signaling pathway in mice.Methods A total of 90 mice were randomly separated into the control group,the cigarette smoke(CS)group,the CA low-dose treatment group(CA-L group),the CA high-dose treatment group(CA-H group)and the CA high-dose treatment plus SIRT3 inhibitor 3-TYP group(CA-H+3-TYP group),with 18 mice in each group.Tidal volume(TV)and peak expiratory flow rate(PEF)of lung function were detected by whole body plethysmography system.Serum levels of inflammatory factors[interleukin(IL)-6,tumor necrosis factor(TNF)-α]and oxidative stress indicators[reactive oxygen species(ROS),SOD]were detected by enzyme-linked immunosorbent assay(ELISA).The injury of airway epithelial cells in lung tissue was observed by HE staining.The expression levels of barrier related proteins(OCLN and ZO-1)in airway epithelial cells were detected by immunohistochemistry.Immunoblotting was applied to detect the expression of SIRT3/SOD2 signaling pathway related proteins.Results Compared with the control group,levels of TV,PEF,MAN and SOD and the expression levels of OCLN,ZO-1,SIRT3 and SOD2 were decreased in the CS group,while the levels of MLI,IL-6,TNF-α and ROS were increased(P＜0.05).Compared with the control group,the lung tissue structure was significantly damaged,the alveolar enlargement was obvious,the surrounding alveolar was accompanied by inflammatory cell infiltration,and the airway epithelial cells were obviously shed in the CS group.Different doses of CA alleviated lung tissue destruction,improved alveolar structure,reduced inflammatory cell infiltration,reduced airway epithelial cell shedding,increased levels of TV,PEF,MAN,SOD and OCLN,ZO-1,SIRT3 and SOD2,and decreased levels of MLI,IL-6,TNF-α and ROS.The effect of high dose CA was more significant than that of low dose CA(P＜0.05).SIRT3/SOD2 signaling pathway inhibitor 3-TYP partially reversed the ameliorative effect of CA on CS induced airway epithelial cell injury in mice.Conclusion CA can ameliorate CS induced airway epithelial cell damage in mice,and its mechanism is related to the activation of the SIRT3/SOD2 signaling pathway.

Objective:To investigate molecular mechanisms underlying the inflammatory response induced by Cutibacterium acnes ( C. acnes) biofilms in human primary keratinocytes. Methods:A C. acnes biofilm model was established in vitro, and confocal fluorescence microscopy was performed to examine its three-dimensional structure. The cultured human primary keratinocytes were divided into 3 groups: a dimethyl sulfoxide (DMSO) control group (treated with 0.01% DMSO alone), a C. acnes suspension group (co-incubated with C. acnes suspensions), and a C. acnes biofilm group (co-incubated with C. acnes biofilms). Real-time fluorescence-based quantitative PCR (RT-qPCR) was performed to determine the relative mRNA expression of interleukin (IL) -6, IL-8, and tumor necrosis factor (TNF) -α in the groups after 6-hour culture, enzyme-linked immunosorbent assay to detect the free protein levels of IL-6, IL-8, and TNF-α in the groups after 24-hour culture, and Western blot analysis to determine the protein expression of Toll-like receptor 2 (TLR2) in keratinocytes. In addition, some human primary keratinocytes were pretreated with key molecular blockers targeting the TLR2/mitogen-activated protein kinase (MAPK) /nuclear factor (NF) -κB signaling pathway (C29, ST2825, BAY11-7082, SB203580, U0126-EtOH), and then co-incubated with C. acnes biofilms; the DMSO control group and the C. acnes biofilm group receiving no pretreatment were simultaneously set as negative and positive controls, respectively. The mRNA and free protein expression levels of IL-6, IL-8, and TNF-α were then detected in the above groups. One-way analysis of variance was used for comparisons among multiple groups, and the Bonferroni method was used for multiple comparisons. Results:Confocal fluorescence microscopy demonstrated a three-dimensional C. acnes biofilm structure resembling a lawn, and the biofilm grew well. RT-qPCR and ELISA showed significant differences in the mRNA and free protein expression levels of IL-6, IL-8, and TNF-α among the C. acnes biofilm group, C. acnes suspension group and DMSO control group (mRNA: F = 89.70, 312.17, 46.09, respectively, all P < 0.001; free protein: F = 886.12, 634.25, 307.01, respectively, all P < 0.001) ; in detail, the mRNA and free protein expression levels of IL-6, IL-8, and TNF-α were significantly higher in the C. acnes biofilm group than in the C. acnes suspension group and DMSO control group (all P < 0.001) ; the C. acnes suspension group showed significantly increased expression levels of IL-6 mRNA and TNF-α free protein compared with the DMSO control group ( P < 0.001, = 0.003, respectively), while there were no significant differences in the expression of IL-6 free protein, TNF-α mRNA, or IL-8 mRNA and free protein between the 2 groups (all P > 0.05). Western blot analysis showed that the TLR2 protein expression was significantly higher in the C. acnes suspension group and C. acnes biofilm group than in the DMSO control group. After the pretreatment with molecular blockers targeting the MAPK/NF-κB signaling pathway and co-incubation with C. acnes biofilms, the mRNA and free protein expression levels of IL-6, IL-8 and TNF-α were all significantly lower in the C29 group, ST2825 group, BAY11-7082 group, SB203580 group, U0126-EtOH group, as well as in the DMSO control group compared with the C. acnes biofilm group (all P < 0.05) . Conclusion:The C. acnes biofilms exhibited a strong ability to induce inflammatory responses in human keratinocytes, possibly through the activation of the TLR2/MAPK/NF-κB signaling pathway.

Objective To observe the effect of Danshen injection (DSI) on pulmonary fibrosis in silicosis mice, and to analyze the differential metabolic pathway on pulmonary fibrosis in silicosis using DSI by urine metabolomics. Methods The specific pathogen free C57BL/6J mice were randomly divided into control group, silicosis model group, DSI prevention group and DSI treatment group. The mice in the last three groups were given 1 mL silica suspension with a mass concentration of 50 g/L by the one-time non-exposed tracheal method, and the mice in the control group were not given any treatment. Subsequently, mice in the DSI prevention group and the DSI treatment group were given intraperitoneal injection of DSI with a dose of 5 mL/kg body weight from 24 hours after exposure to dust and from the 29th day after exposure to dust, respectively, once per day until the 56th day after exposure. Mice in the other two groups were not treated. After DSI intervention, the lung histopathological changes of mice in all groups were evaluated. The components of mouse urine metabolites were analyzed using ultra-high performance liquid chromatography-quadrupole-time-of-fight mass spectrometry method. Human Metabolome Database was used to screen the potential differential metabolites (DMs). The related metabolic pathways were analyzed using MetaboAnanlyst 5.0 Web analytics platform. Results The result of hematoxylin-eosin staining and Van Gieson staining of mouse lung tissues showed that the pulmonary alveolar structure destroyed, typical fibrotic nodules appeared, collagen fiber deposition increased, and clumpy accumulation in the silicosis model group, compared with the control group. Compared with the silicosis model group, the degree of pulmonary alveolar inflammation and fibrosis in the lung tissues of mice in the DSI prevention group was obviously reduced to close to the control group, while pulmonary alveolar inflammation and fibrosis in the lung tissues of mice in the DSI treatment group were also reduced, although the outcome was not as good as that in the DSI prevention group. The result of urine metabolomics analysis identified four DMs in the model group and control group, seven DMs were identified in the DSI prevention group and silicosis model group, seven DMs were identified in the DSI treatment group and silicosis model group. A total of three DMs pathways related to pulmonary fibrosis in silicosis model group and the protective effect of DSI prevention group were identified, including D-arginine and D-ornithine metabolism, folic acid biosynthesis and metabolism, pantothenate and succinyl coenzyme A biosynthesis pathways (all P<0.01). Conclusion DSI treatment in any time point can interfere the process of pulmonary fibrosis in the silicosis mice, while the interference is more effective in the DSI group treated right after dust-exposure. DSI interferes with the urinary metabolism pathway of silicosis mice, and the D-arginine and D-ornithine metabolism, folic acid biosynthesis and metabolism, pantothenate and succinyl coenzyme A biosynthesis pathways may participate in the inhibiting process of early pulmonary fibrosis in silicosis mice by DSI.