BACKGROUND Chronic inflammation due to Helicobacter pylori(H.pylori)infection promotes gastric carcinogenesis.Tumour necrosis factor-α(TNF-α),a key mediator of inflammation,induces cell survival or apoptosis by bind...BACKGROUND Chronic inflammation due to Helicobacter pylori(H.pylori)infection promotes gastric carcinogenesis.Tumour necrosis factor-α(TNF-α),a key mediator of inflammation,induces cell survival or apoptosis by binding to two receptors(TNFR1 and TNFR2).TNFR1 can induce both survival and apoptosis,while TNFR2 results only in cell survival.The dysregulation of these processes may contribute to carcinogenesis.AIM To evaluate the effects of TNFR1 and TNFR2 downregulation in AGS cells treated with H.pylori extract on the TNF-αpathway.METHODS AGS cell lines containing TNFR1 and TNFR2 receptors downregulated by specific shRNAs and nonsilenced AGS cells were treated with H.pylori extract for 6 h.Subsequently,quantitative polymerase chain reaction with TaqMan®assays was used for the relative quantification of the mRNAs(TNFA,TNFR1,TNFR2,TRADD,TRAF2,CFLIP,NFKB1,NFKB2,CASP8,CASP3)and miRNAs(miR-19a,miR-34a,miR-103a,miR-130a,miR-181c)related to the TNF-αsignalling pathway.Flow cytometry was employed for cell cycle analysis and apoptosis assays.RESULTS In nonsilenced AGS cells,H.pylori extract treatment increased the expression of genes involved in cell survival and inhibited both apoptosis(NFKB1,NFKB2 and CFLIP)and the TNFR1 receptor.TNFR1 downregulation significantly decreased the expression of the TRADD and CFLIP genes,although no change was observed in the cellular process or miRNA expression.In contrast,TNFR2 downregulation decreased the expression of the TRADD and TRAF2 genes,which are both important downstream mediators of the TNFR1-mediated pathway,as well as that of the NFKB1 and CFLIP genes,while upregulating the expression of miR-19a and miR-34a.Consequently,a reduction in the number of cells in the G0/G1 phase and an increase in the number of cells in the S phase were observed,as well as the promotion of early apoptosis.CONCLUSION Our findings mainly highlight the important role of TNFR2 in the TNF-αpathway in gastric cancer,indicating that silencing it can reduce the expression of survival and anti-apoptotic genes.展开更多
Objective:To investigate the protective effect of Chinese herbal formula Huangqin Decoction(HQD)on ulcerative colitis mouse model induced by dextran sulphate sodium(DSS)and human intestinal epithelial cell injury indu...Objective:To investigate the protective effect of Chinese herbal formula Huangqin Decoction(HQD)on ulcerative colitis mouse model induced by dextran sulphate sodium(DSS)and human intestinal epithelial cell injury induced by tumour necrosis factor-α(TNF-α).Methods:In vivo,30 male C57BL/6 mice were divided into 5 groups using a random number table(n=6 per group),including control,DSS,5-aminosalicylic acid(5-ASA),HQD low-(HQD-L)and high-dose(HQD-H)groups.The colitis mouse model was established by 3%(w/v)DSS water for 5 days.Meanwhile,mice in the HQD-L,HQD-H and 5-ASA groups were administrated with 100,200 mg/kg HQD or 100 mg/kg 5-ASA,respectively,once daily by gavage.After 9 days of administration,the body weight,disease activity index(DAI)score and colon length of mice were measured,the pathological changes of colons were analyzed by hematoxylin-eosin staining(HE)staining,and the levels of serum interleukin(IL)-6,IL-1βand TNF-αwere measured by enzyme linked immunosorbent assay.In vitro,the human colon epithelial normal cells(FHC cells)were exposed to HQD(0.6 mg/mL)for 12 h and then treated with TNF-α(10 ng/mL)for 24 h.The tight junction(TJ)protein expression levels of Claudin-4 and Occludin,and the protein phosphorylation levels of p65 and inhibitor of nuclear factor kappaB(NF-κB)-α(IκBα)were measured by Western blot.Results:In vivo,compared with the DSS group,HQD-H treatment attenuated the weight loss and reduced DAI score of mice on the 8th day(P<0.05).Moreover,HQD-H treatment ameliorated the colon shortening in the DSS-induced colitis mice(P<0.05).HE staining showed HQD attenuated the pathological changes of colitis mice,and the histological scores of HQD-H and 5-ASA groups were significantly decreased compared with the DSS group(P<0.05).Meanwhile,HQD-H and 5-ASA significantly decreased the serum IL-1β,IL-6 and TNF-αlevels of mice(P<0.05).In vitro experiments showed that HQD up-regulated Occludin and Claudin-4 protein expressions and inhibited p-p65 and p-IκBαlevels in FHC cells compared with the TNF-αgroup(P<0.05).Conclusion:HQD significantly relieved the symptoms in DSS-induced colitis mice by inhibiting pro-inflammatory cytokines expression and maintained the homeostasis of TJ protein in FHC cells by suppressing TNF-α-induced NF-κB activation.展开更多
基金Supported by São Paulo Research Foundation(FAPESP),No.2015/21464-0 and No.2015/23392-7National Counsel of Technological and Scientific Development(CNPq),No.310120/2015-2.
文摘BACKGROUND Chronic inflammation due to Helicobacter pylori(H.pylori)infection promotes gastric carcinogenesis.Tumour necrosis factor-α(TNF-α),a key mediator of inflammation,induces cell survival or apoptosis by binding to two receptors(TNFR1 and TNFR2).TNFR1 can induce both survival and apoptosis,while TNFR2 results only in cell survival.The dysregulation of these processes may contribute to carcinogenesis.AIM To evaluate the effects of TNFR1 and TNFR2 downregulation in AGS cells treated with H.pylori extract on the TNF-αpathway.METHODS AGS cell lines containing TNFR1 and TNFR2 receptors downregulated by specific shRNAs and nonsilenced AGS cells were treated with H.pylori extract for 6 h.Subsequently,quantitative polymerase chain reaction with TaqMan®assays was used for the relative quantification of the mRNAs(TNFA,TNFR1,TNFR2,TRADD,TRAF2,CFLIP,NFKB1,NFKB2,CASP8,CASP3)and miRNAs(miR-19a,miR-34a,miR-103a,miR-130a,miR-181c)related to the TNF-αsignalling pathway.Flow cytometry was employed for cell cycle analysis and apoptosis assays.RESULTS In nonsilenced AGS cells,H.pylori extract treatment increased the expression of genes involved in cell survival and inhibited both apoptosis(NFKB1,NFKB2 and CFLIP)and the TNFR1 receptor.TNFR1 downregulation significantly decreased the expression of the TRADD and CFLIP genes,although no change was observed in the cellular process or miRNA expression.In contrast,TNFR2 downregulation decreased the expression of the TRADD and TRAF2 genes,which are both important downstream mediators of the TNFR1-mediated pathway,as well as that of the NFKB1 and CFLIP genes,while upregulating the expression of miR-19a and miR-34a.Consequently,a reduction in the number of cells in the G0/G1 phase and an increase in the number of cells in the S phase were observed,as well as the promotion of early apoptosis.CONCLUSION Our findings mainly highlight the important role of TNFR2 in the TNF-αpathway in gastric cancer,indicating that silencing it can reduce the expression of survival and anti-apoptotic genes.
基金the Scientific Research Project of Jiangsu Provincial Administration of Traditional Chinese Medicine(No.JD2019SZXYB05)Natural Science Foundation of Nanjing University of Chinese Medicine(No.XZR2020030)National Administration of Traditional Chinese Medicine:2019 Project of Building Evidence-Based Practice Capacity for Traditional Chinese Medicine(No.2019XZZX-XH007)。
文摘Objective:To investigate the protective effect of Chinese herbal formula Huangqin Decoction(HQD)on ulcerative colitis mouse model induced by dextran sulphate sodium(DSS)and human intestinal epithelial cell injury induced by tumour necrosis factor-α(TNF-α).Methods:In vivo,30 male C57BL/6 mice were divided into 5 groups using a random number table(n=6 per group),including control,DSS,5-aminosalicylic acid(5-ASA),HQD low-(HQD-L)and high-dose(HQD-H)groups.The colitis mouse model was established by 3%(w/v)DSS water for 5 days.Meanwhile,mice in the HQD-L,HQD-H and 5-ASA groups were administrated with 100,200 mg/kg HQD or 100 mg/kg 5-ASA,respectively,once daily by gavage.After 9 days of administration,the body weight,disease activity index(DAI)score and colon length of mice were measured,the pathological changes of colons were analyzed by hematoxylin-eosin staining(HE)staining,and the levels of serum interleukin(IL)-6,IL-1βand TNF-αwere measured by enzyme linked immunosorbent assay.In vitro,the human colon epithelial normal cells(FHC cells)were exposed to HQD(0.6 mg/mL)for 12 h and then treated with TNF-α(10 ng/mL)for 24 h.The tight junction(TJ)protein expression levels of Claudin-4 and Occludin,and the protein phosphorylation levels of p65 and inhibitor of nuclear factor kappaB(NF-κB)-α(IκBα)were measured by Western blot.Results:In vivo,compared with the DSS group,HQD-H treatment attenuated the weight loss and reduced DAI score of mice on the 8th day(P<0.05).Moreover,HQD-H treatment ameliorated the colon shortening in the DSS-induced colitis mice(P<0.05).HE staining showed HQD attenuated the pathological changes of colitis mice,and the histological scores of HQD-H and 5-ASA groups were significantly decreased compared with the DSS group(P<0.05).Meanwhile,HQD-H and 5-ASA significantly decreased the serum IL-1β,IL-6 and TNF-αlevels of mice(P<0.05).In vitro experiments showed that HQD up-regulated Occludin and Claudin-4 protein expressions and inhibited p-p65 and p-IκBαlevels in FHC cells compared with the TNF-αgroup(P<0.05).Conclusion:HQD significantly relieved the symptoms in DSS-induced colitis mice by inhibiting pro-inflammatory cytokines expression and maintained the homeostasis of TJ protein in FHC cells by suppressing TNF-α-induced NF-κB activation.
