姜黄素通过阻断NF-κB减少IL-1β诱导的内皮脂酶表达

目的观察姜黄素对培养的人血管内皮细胞内皮脂酶表达的影响,并探讨其可能的作用机制。方法不同浓度的姜黄素处理HUVEC-12细胞。RT-PCR检测内皮酯酶(endo-thelial lipase,EL)mRNA的表达;Western blot检测核因子-κB抑制因子-α(inhibitor of nuclear factor-κB-α,IκB-α)蛋白的表达;间接免疫荧光检测核因子-κB(nuclear factor-κB,NF-κB)蛋白的活化。结果IL-1β处理HUVEC-12细胞可以明显上调EL mRNA的表达,同时降低胞质蛋白IκB-α的表达水平,激活核转录因子NF-κB,增加胞核蛋白NF-κB的水平。姜黄素预处理HUVEC-12细胞可以抑制IL-1β对EL的上调作用,同时逆转IL-1β诱导的IκB-α蛋白降解和NF-κB活化。结论姜黄素可以通过阻断NF-κB活化减少IL-1β诱导的人血管内皮细胞EL的表达。

Inflammatory Mechanism of Total Flavonoids of Chrysanthemum and Medicated Serum on Castrated Dry Eye Animal and Cell Models

Objective To observe the effects of total flavonoids of chrysanthemum and medicated serum on the expression of related proteins in the lacrimal tissue and dry-eye cell models of male rabbits with dry eye caused by castration.Methods(1)150 male Japanese rabbits were randomly divided into five groups,with 30 rabbits in each group:normal control group(group A),sham group(group B),model group(group C),androgen control group(group D)and total flavonoids of chrysanthemum treatment group(group E).The androgen deficiency dry-eye model was established by bilateral castration in groups C,D and E.Normal saline was administered to groups A,B and C by gavage;androgen(testosterone propionate)was injected into muscle in group D;and group E was given total flavonoids of chrysanthemum by gavage.All white rabbits were tested the Schirmer I test(SIT)and tear break-up time(BUT).After euthanasia,tear gland tissue was harvested so that we could observe pathological changes in the expression of related inflammatory factors in the lacrimal gland tissue.The expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and transforming growth factor-β1(TGF-β1)was detected in the lacrimal gland tissue by immunohistochemistry.Reverse transcription PCR was used to quantitatively detect expression of TGF-β1 mRNA.(2)Male Wistar rat lacrimal epithelial cells were used to establish a model of eye stem cell apoptosis caused by androgen levels.The blank control group was set up without androgen culture,the control group with androgen culture,and the total flavonoids of chrysanthemum group without androgen.The MTT method was used to determine the optimal intervention dosage of drug-containing plasma.Western blot and QPCR were used to detect the expression of AR mRNA,NF-κB phosphorylated protein and TGF-β1 in lacrimal epithelial cells,and the androgen-like effect of total flavonoids of chrysanthemum was observed.Results(1)Immunohistochemistry showed that groups A,B,D and E had significantly lower expression of IL-1βand TNF-αthan group C(P<0.05);among these,group E had slightly higher expression than group D(P>0.05).RT-PCR results showed that the relative expression of TGF-β1 mRNA in groups A,B,D and E was significantly higher than in group C(P<0.05),and the relative expression of TGF-β1 mRNA in groups D and E was higher than that in groups A and B(P<0.05).(2)Using the MTT method,the final concentration of interfering cells was calculated to be 13.2%.The expression of AR protein,NF-κB and TGF-β1 in the chrysanthemum flavonoid plasma intervention and testosterone propionate intervention groups was enhanced,and there were significant differences relative to the blank group(P<0.01).The expression level of NF-κB in the total flavonoid containing plasma intervention group was lower than that in the testosterone propionate intervention group(P<0.01).Conclusions The total flavonoids of chrysanthemum can inhibit IL-1βand TNF-αexpression in the lacrimal gland tissue of castrated male rabbits with dry eye to increase synthesis of TGF-β1 mRNA and TGF-β1,thereby inhibiting the inflammatory response.The medicated plasma with total flavonoids of chrysanthemum promotes expression of AR mRNA,upregulating expression of NF-κB,further promoting upregulation of TGF-β1 protein expression in lacrimal epithelial cells,inhibiting inflammation by regulating related proteins,and ultimately alleviating the symptoms of dry eye.

Effect of cefodizime on cytokines expression in mouse neutrophils and its related mechanism

Objective： To explore the regulating effects of cefodizime on cytokines expression of neutrophil response to Klebsiella pneumoniae （Kle. p） treatment. Methods： We detected the types and expression of cytokines secreted by neutrophils by cDNA array and RT-PCR. We also analyzed the changes of signal transduction in this process by detecting the expression of toll like receptor 4 （TLR4） and the inhibitor factor of κBα （I-κBα） expressed by neutrophils. The activity of NF-κB DNA-binding in neutrophils was measured by electrophoretic mobility shift assay （EMSA）. Results： Cefodizime increased the neutrophils production of TNF-α, IL-β3 and the mRNA expression of TLR4 in the early stage of Kle. p stimulation in mice, which seemed corresponding to the enhanced NF-κB DNA-binding activity. Conclusion： Cefodizime regulates the cytokines expression of neutrophils through the LPS-TLR4-NF-κB pathway by affecting the expression of TLR4 mRNA and the DNA binding activities of NF-κB in mice with the challenge of Kle. p.

Regulation of CD137 expression through K-Ras signaling in pancreatic cancer cells

Background:The interaction between CD137 and its ligand(CD137L)plays a major role in the regulation of immune functions and affects cancer immunotherapy.CD137 is a cell surface protein mainly located on activated T cells,and its regulation and functions in immune cells are well established.However,the expression of CD137 and its regulation in cancer cells remain poorly understood.The main purposes of this study were to examine the expression of CD137 in pancreatic cancer cells and to investigate its underlying mechanisms.Methods:Cells containing inducible K-RasG12V expression vector or with different K-Ras mutational statuses were used as in vitro models to examine the regulation of CD137 expression by K-Ras.Various molecular assays were employed to explore the regulatory mechanisms.Tumor specimens from 15 pancreatic cancer patients and serum samples from 10 patients and 10 healthy donors were used to test if the expression of CD137 could be validated in clinical samples.Results:We found that the CD137 protein was expressed on the cell surface in pancreatic cancer tissues and cancer cell lines.Enzyme-linked immunosorbent assay revealed no difference in the levels of secreted CD137 in the sera of patients and healthy donors.By using the K-Ras inducible cell system,we further showed that oncogenic K-Ras up-regulated CD137 through the activation of MAPK(mitogen-activated protein kinases)and NF-κB(nuclear factor kappa-light-chain-enhancer of activated B cells)pathways,as evidenced by significantly reduced CD137 mRNA expression led by genetic silencing of MAPK1 and p65,the key proteins involved in the respective pathways.Further-more,we also found that the NF-κB pathway was mainly stimulated by the K-Ras-induced secretion of interleukin-1α(IL-1α)which promoted the transcription of the CD137 gene in pancreatic cancer cell lines.Analysis of the TCGA(the cancer genome atlas)database also revealed a significant correlation between IL-1αand CD137 expression(r=0.274)in tumor samples from pancreatic cancer patients(P<0.001).Conclusions:The present study has demonstrated that the CD137 protein was expressed on pancreatic cancer cell surface,and has identified a novel mechanism by which K-Ras regulates CD137 in pancreatic cancer cells through MAPK and NF-κB pathways stimulated by IL-1α.