背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供...背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。展开更多
Previous studies have suggested that polypeptides extracted from milk, soybean, fish, eggs, and meat possess potential anti-inflammatory effects. To date, few studies have reported the anti-inflammatory function of st...Previous studies have suggested that polypeptides extracted from milk, soybean, fish, eggs, and meat possess potential anti-inflammatory effects. To date, few studies have reported the anti-inflammatory function of sturgeon peptides and their underlying mechanisms are unknown. The current study was therefore to determine the anti-inflammatory potential of sturgeon peptides with lipopolysaccharide (LPS)-induced RAW264.7 inflammatory model. Pepsin hydrolysate (PeH) was purified by ultrafiltration and Sephadex G-15 gel filtration chromatography. PeH significantly reduced the inflammatory mediator (NO) and inflammatory cytokines (IL-6, TNF-α and IL-1β) expression in a dose-dependent manner. Moreover, the purified sturgeon peptide (F2) possessed strong antioxidant potential and effectively inhibited DPPH and ABTS free radicals. F2 significantly suppressed the expression of MAPK, IκBα, and NF-κB p65, indicating that F2 exerted anti-inflammatory influence by the inhibition of MAPK and NF-κB pathways.展开更多
Objective:To explore the anti-inflammatory effect of the traditional Chinese medicine Zhikang capsule(ZKC)on lipopolysaccharide(LPS)-induced RAW264.7 cells.Methods:Safe concentrations of ZKC(0.175,0.35,and 0.7 mg/mL)w...Objective:To explore the anti-inflammatory effect of the traditional Chinese medicine Zhikang capsule(ZKC)on lipopolysaccharide(LPS)-induced RAW264.7 cells.Methods:Safe concentrations of ZKC(0.175,0.35,and 0.7 mg/mL)were used after the half-maximal inhibitory concentration(IC_(50))of RAW264.7 cells was calculated through the CCK-8 assay.In addition,the optimal intervention duration of ZKC(0.7 mg/mL)on RAW264.7 cells was determined to be 6 h,since all proinflammatory mediators[tumor necrosis factor-alpha(TNF-α),interleukin-1 beta(IL-1β),inteleukin-6(IL-6),cyclooxygenase-2(COX-2),inducible nitric oxide synthase(iNOS),and monocyte chemotactic protein-1(MCP-1)]had a decreasing tendency and relatively down-regulated mRNA expression levels as compared with other durations(4,8,and 12 h).RAW264.7 cells were pretreated with ZKC at various concentrations(0.175,0.35 and 0.7 mg/mL)for 6 h and then stimulated with LPS(1 μg/mL)for an additional 12 h.Results:In terms of inflammation,ZKC could reverse LPS-induced upregulation of TNF-α,IL-1β,IL-6,COX-2,iNOS,and MCP-1 at both the mRNA and protein levels in RAW264.7 cells in a dose-dependent manner.In terms of the NF-κB signaling pathway,ZKC could reduce phosphorylated p65 and promote M2 polarization of RAW264.7 cells under LPS stimulation in a dose-dependent manner.Moreover,ZKC exhibited a protective effect on macrophages from apoptosis.Conclusion:ZKC exhibited obvious antiinflammatory and anti-apoptotic effects on LPS-induced RAW264.7 cells at the cellular level,and a weakened NF-κB signaling pathway may be a potential significant target.展开更多
It has been observed that the consumption of litchi often causes symptoms characterized by itching or sore throat,gum swelling,oral cavity ulcers and even fever and inflammation,which significantly impair the quality ...It has been observed that the consumption of litchi often causes symptoms characterized by itching or sore throat,gum swelling,oral cavity ulcers and even fever and inflammation,which significantly impair the quality of life of a large population.Using the RAW264.7 cell line,a step-by-step strategy was used to screen for the components in litchi fruits that elicited adverse reactions.The adverse reaction fractions were identified by mass spectrometry and analyzed using the SMART program,and a sequence alignment of the homologous proteins was performed.MTT tests were used to determine the cytotoxicity of a litchi protein extract in RAW264.7 macrophages,and real-time PCR was applied to analyze the expression of inflammatory genes in the RAW264.7 cells treated with lipopolysaccharide or the litchi protein extract.The results showed that the litchi water-soluble protein extract could increase the production of the pro-inflammatory mediators IL-1β,iNOS and COX-2,and the anti-inflammatory mediator HO-1 in the RAW264.7 cell line.The 14-3-3-like proteins GF14 lambda,GF14 omega and GF14 upsilon were likely the candidate proteins that caused the adverse effects.展开更多
Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide ...Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.展开更多
文摘Previous studies have suggested that polypeptides extracted from milk, soybean, fish, eggs, and meat possess potential anti-inflammatory effects. To date, few studies have reported the anti-inflammatory function of sturgeon peptides and their underlying mechanisms are unknown. The current study was therefore to determine the anti-inflammatory potential of sturgeon peptides with lipopolysaccharide (LPS)-induced RAW264.7 inflammatory model. Pepsin hydrolysate (PeH) was purified by ultrafiltration and Sephadex G-15 gel filtration chromatography. PeH significantly reduced the inflammatory mediator (NO) and inflammatory cytokines (IL-6, TNF-α and IL-1β) expression in a dose-dependent manner. Moreover, the purified sturgeon peptide (F2) possessed strong antioxidant potential and effectively inhibited DPPH and ABTS free radicals. F2 significantly suppressed the expression of MAPK, IκBα, and NF-κB p65, indicating that F2 exerted anti-inflammatory influence by the inhibition of MAPK and NF-κB pathways.
文摘Objective:To explore the anti-inflammatory effect of the traditional Chinese medicine Zhikang capsule(ZKC)on lipopolysaccharide(LPS)-induced RAW264.7 cells.Methods:Safe concentrations of ZKC(0.175,0.35,and 0.7 mg/mL)were used after the half-maximal inhibitory concentration(IC_(50))of RAW264.7 cells was calculated through the CCK-8 assay.In addition,the optimal intervention duration of ZKC(0.7 mg/mL)on RAW264.7 cells was determined to be 6 h,since all proinflammatory mediators[tumor necrosis factor-alpha(TNF-α),interleukin-1 beta(IL-1β),inteleukin-6(IL-6),cyclooxygenase-2(COX-2),inducible nitric oxide synthase(iNOS),and monocyte chemotactic protein-1(MCP-1)]had a decreasing tendency and relatively down-regulated mRNA expression levels as compared with other durations(4,8,and 12 h).RAW264.7 cells were pretreated with ZKC at various concentrations(0.175,0.35 and 0.7 mg/mL)for 6 h and then stimulated with LPS(1 μg/mL)for an additional 12 h.Results:In terms of inflammation,ZKC could reverse LPS-induced upregulation of TNF-α,IL-1β,IL-6,COX-2,iNOS,and MCP-1 at both the mRNA and protein levels in RAW264.7 cells in a dose-dependent manner.In terms of the NF-κB signaling pathway,ZKC could reduce phosphorylated p65 and promote M2 polarization of RAW264.7 cells under LPS stimulation in a dose-dependent manner.Moreover,ZKC exhibited a protective effect on macrophages from apoptosis.Conclusion:ZKC exhibited obvious antiinflammatory and anti-apoptotic effects on LPS-induced RAW264.7 cells at the cellular level,and a weakened NF-κB signaling pathway may be a potential significant target.
基金This work was supported by the Special Fund for Agro-scientific Research in the Public Interest(No.201403036)the Fundamental Research Funds for the Central Universities(2014PY030)+1 种基金the National Basic Research Program of China(2011CB100600)the National NSF of China and the Foundation for the Key Program of the Ministry of Education,China(311029).
文摘It has been observed that the consumption of litchi often causes symptoms characterized by itching or sore throat,gum swelling,oral cavity ulcers and even fever and inflammation,which significantly impair the quality of life of a large population.Using the RAW264.7 cell line,a step-by-step strategy was used to screen for the components in litchi fruits that elicited adverse reactions.The adverse reaction fractions were identified by mass spectrometry and analyzed using the SMART program,and a sequence alignment of the homologous proteins was performed.MTT tests were used to determine the cytotoxicity of a litchi protein extract in RAW264.7 macrophages,and real-time PCR was applied to analyze the expression of inflammatory genes in the RAW264.7 cells treated with lipopolysaccharide or the litchi protein extract.The results showed that the litchi water-soluble protein extract could increase the production of the pro-inflammatory mediators IL-1β,iNOS and COX-2,and the anti-inflammatory mediator HO-1 in the RAW264.7 cell line.The 14-3-3-like proteins GF14 lambda,GF14 omega and GF14 upsilon were likely the candidate proteins that caused the adverse effects.
基金supported by the National Natural Science Foundation of China,No.32371048(to YK)the Peking University People’s Hospital Research and Development Funds,No.RDX2021-01(to YK)the Natural Science Foundation of Beijing,No.7222198(to NH)。
文摘Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.
文摘为解析槲皮万寿菊素、槲皮素与叶黄素单独处理以及联合处理对急性肺损伤的作用机制,以脂多糖诱导构建RAW264.7细胞炎症模型,以一氧化氮(nitric oxide,NO)相对含量为评价指标,采用联合指数法确定槲皮万寿菊素与叶黄素以及槲皮素与叶黄素的最佳复配比例;分析比较槲皮万寿菊素、槲皮素与叶黄素单独及联合处理对RAW264.7细胞中炎症因子(肿瘤坏死因子-α、白细胞介素(interleukin,IL)-1β、IL-6)含量、丙二醛含量、超氧化物歧化酶活性以及谷胱甘肽过氧化物酶活性的影响;采用免疫印迹法测定核因子κB(nuclear factor kappa-B,NF-κB)信号通路中p65、p50以及沉默信息调节因子1(silent information regulator 1,SIRT1)、核因子E2相关因子2(nuclear factor-erythroid 2 related factor 2,Nrf2)和NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)的相对表达量。结果表明,槲皮万寿菊素与叶黄素高剂量3∶1(30μg/mL+10μg/mL)复配能够最大程度降低RAW264.7细胞中的NO相对含量。二者单独及联合作用均能通过降低炎症因子、丙二醛含量,提高超氧化物歧化酶和谷胱甘肽还原酶活性,下调NF-κB p65、p50以及NLRP3表达水平并上调SIRT1、Nrf2蛋白相对表达量发挥改善急性肺损伤的作用,且联合处理效果优于单独处理组。
