小檗碱干预高糖环境下人肝癌细胞株HCCLM3细胞的作用研究
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摘要
目的观察小檗碱对高糖环境下人肝癌细胞株HCCLM3细胞增殖的抑制、自噬作用。方法将传至3—4代HCCLM3细胞随机分为空白对照组和低糖组、中糖组、高糖组(各组分别加入含有1000、2000、4000、8000 mg·L~(-1)的葡萄糖培养液)及小檗碱A组、小檗碱B组、小檗碱C组、二甲双胍(MET)组(高糖组不给予任何干预措施,小檗碱A、B、C 3组分别加入小檗碱含药培养液5、10、20μmol·L~(-1),MET组加入MET含药培养液10μmol·L~(-1))。CCK-8检测HCCLM3细胞的增殖率、增殖抑制率,酶联免疫吸附试验法(ELISA)检测HCCLM3细胞上清液IL-6、TNF-α的水平,实时荧光定量PCR (qPCR)检测HCCLM3细胞NF-κB mRNA、Beclin1 mRNA的表达水平。结果中糖、高糖2组24 h时HCCLM3细胞增殖率均高于空白对照组(均P<0.05)。小檗碱A、B、C 3组和MET组24、48、72 h时HCCLM3细胞增殖抑制率均高于高糖组(均P<0.05),小檗碱A、B、C 3组24、48、72 h时HCCLM3细胞增殖抑制率均高于MET组(均P<0.05)。小檗碱A、B、C 3组和MET组HCCLM3细胞上清液IL-6、TNF-α水平及HCCLM3细胞NF-κB mRNA表达水平均低于高糖组(均P<0.01),小檗碱A、B 2组HCCLM3细胞上清液IL-6、TNF-α水平均高于MET组(均P<0.05),小檗碱A组HCCLM3细胞NF-κB mRNA表达水平高于MET组、Beclin1 mRNA表达水平低于MET组(均P<0.01),小檗碱B、C 2组HCCLM3细胞Beclin1 mRNA表达水平均低于MET组、小檗碱C组HCCLM3细胞上清液TNF-α水平低于MET组(P<0.05或P<0.01)。结论 BBR对高糖环境下HCCLM3细胞增殖具有抑制作用,它可能是通过调控核因子NF-κB降低IL-6、TNF-α的表达,进而上调Beclin1基因表达水平,促进自噬产生抗肿瘤作用。
Objective To explore the effect of berberine on proliferation and autophagy in hepatocellular carcinoma HCCLM3 cells under high glucose condition.Methods HCCLM3 cells at passages 3-4 were randomly cultured in medium supplemented with glucose at 1000(blank control group),2000(low glucose group),4000(middle glucose group) or 8000 mg·L~(-1)(high glucose group).In addition,cells cultured under high glucose condition were treated with 5 μmol·L~(-1) berberine(berberine A group),10 μmol/L berberine(berberine B group),20 μmol/L berberine(berberine C group) or 10 μmol/L metformin(MET group).The proliferation of cells was detected by CCK-8 assay.The levels of interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in supernatant were measured by ELISA.The expression of NF-κB and Beclin1 mRNA was determined by real-time quantitative PCR.Results The proliferation rate of HCCLM3 cells in middle glucose or high glucose group was higher than that in blank control group after treatment for 24 hours(P<0.05).After treatment for 24,48 and 72 hours,the proliferation inhibition rate of HCCLM3 cells in berberine A,berberine B,berberine C or MET group was higher than that in high glucose group(P<0.05),and that in berberine A,B or C group was higher than that in MET group(P<0.05).The levels of IL-6 and TNF-α in supernatant and the expression of NF-κB mRNA in HCCLM3 cells in berberine groups and MET group were lower than those in high glucose group(P<0.01).The levels of IL-6 and TNF-α in supernatant in berberine A or B group were higher than those in MET group(P<0.05).However,the levels of TNF-α in berberine C group were lower than those in MET group(P<0.05).The expression of NF-κB mRNA in HCCLM3 cells in berberine A group was higher than that in MET group(P<0.01).The expression of Beclin1 mRNA in HCCLM3 cells in all the three berberine groups was lower than that in MET group(P<0.05 or P<0.01).Conclusion Berberine could inhibit the proliferation of HCCLM3 cells under high glucose environment through regulating NF-κB expression to reduce IL-6 and TNF-α production and up-regulating Beclin1 expression to promote autophagy.
Objective To explore the effect of berberine on proliferation and autophagy in hepatocellular carcinoma HCCLM3 cells under high glucose condition.Methods HCCLM3 cells at passages 3-4 were randomly cultured in medium supplemented with glucose at 1000(blank control group),2000(low glucose group),4000(middle glucose group) or 8000 mg·L~(-1)(high glucose group).In addition,cells cultured under high glucose condition were treated with 5 μmol·L~(-1) berberine(berberine A group),10 μmol/L berberine(berberine B group),20 μmol/L berberine(berberine C group) or 10 μmol/L metformin(MET group).The proliferation of cells was detected by CCK-8 assay.The levels of interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in supernatant were measured by ELISA.The expression of NF-κB and Beclin1 mRNA was determined by real-time quantitative PCR.Results The proliferation rate of HCCLM3 cells in middle glucose or high glucose group was higher than that in blank control group after treatment for 24 hours(P<0.05).After treatment for 24,48 and 72 hours,the proliferation inhibition rate of HCCLM3 cells in berberine A,berberine B,berberine C or MET group was higher than that in high glucose group(P<0.05),and that in berberine A,B or C group was higher than that in MET group(P<0.05).The levels of IL-6 and TNF-α in supernatant and the expression of NF-κB mRNA in HCCLM3 cells in berberine groups and MET group were lower than those in high glucose group(P<0.01).The levels of IL-6 and TNF-α in supernatant in berberine A or B group were higher than those in MET group(P<0.05).However,the levels of TNF-α in berberine C group were lower than those in MET group(P<0.05).The expression of NF-κB mRNA in HCCLM3 cells in berberine A group was higher than that in MET group(P<0.01).The expression of Beclin1 mRNA in HCCLM3 cells in all the three berberine groups was lower than that in MET group(P<0.05 or P<0.01).Conclusion Berberine could inhibit the proliferation of HCCLM3 cells under high glucose environment through regulating NF-κB expression to reduce IL-6 and TNF-α production and up-regulating Beclin1 expression to promote autophagy.
引文
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[2]CAPECE D,VERZELLA D,TESSITORE A,et al.Cancer secretome and inflammation:the bright and the dark sides of NF-κB[J].Semin Cell Dev Biol,2018,78:51-61.
[3]HUA F,YU J J,HU Z W.Diabetes and cancer,common threads and missing links[J].Cancer Lett,2016,374(1):54-61.
[4]LIU C S,ZHENG Y R,ZHANG Y F,et al.Research progress on berberine with a special focus on its oral bioavailability[J].Fitoterapia,2016,109:274-282.
[5]LI Z,GENG Y N,JIANG J D,et al.Antioxidant and anti-inflammatory activities of berberine in the treatment of diabetes mellitus[J].Evid Based Complement Alternat Med,2014,2014:289264.
[6]POLLAK M.The effects of metformin on gut microbiota and the immune system as research frontiers[J].Diabetologia,2017,60(9):1662-1667.
[7]SO S′NICKI S,KAPRAL M,WGLARZ L.Molecular targets of metformin antitumor action[J].Pharmacol Rep,2016,68(5):918-925.
[8]HEALY M E,LAHIRI S,HARGETT S R,et al.Dietary sugar intake increases liver tumor incidence in female mice[J].Sci Rep,2016,6:22292.
[9]LOFTFIELD E,FREEDMAN N D,LAI G Y,et al.Higher glucose and insulin levels are associated with risk of liver cancer and chronic liver disease mortality among men without a history of diabetes[J].Cancer Prev Res(Phila),2016,9(11):866-874.
[10]ROUJUN C,YANHUA Y,BIXUN L.High prevalence of diabetes mellitus and impaired glucose tolerance in liver cancer patients:a hospital based study of 4610patients with benign tumors or specific cancers[J].F1000Res,2016,5:1397.
[11]VASCONCELOS DOSSANTOS A,DE QUEIROZ R M,DACOSTA RODRIGUES B,et al.Hyperglycemia and aberrant O-GlcNAcylation:contributions to tumor progression[J].JBioenerg Biomembr,2018,50(3):175-187.
[12]CHANG S C,YANG W V.Hyperglycemia,tumorigenesis,and chronic inflammation[J].Crit Rev Oncol Hematol,2016,108:146-153.
[13]WANG X,HRING M F,RATHJEN T,et al.Insulin resistance in vascular endothelial cells promotes intestinal tumour formation[J].Oncogene,2017,36(35):4987-4996.
[14]ZHANG H L,WANG M D,ZHOU X,et al.Blocking preferential glucose uptake sensitizes liver tumor-initiating cells to glucose restriction and sorafenib treatment[J].Cancer Lett,2017,388:1-11.
[15]KERN P A,RANGANATHA N S,LI C,et al.Adipose tissue tumor necrosis factor and interleukin-6expression in human obesity and insulin resistance[J].Am J Physiol Endocrinol Metab,2001,280(5):E745-E751.
[16]CHEN S,YANG T,LIU F,et al.Inflammatory factor-specific sumoylation regulates NF-κB signalling in glomerular cells from diabetic rats[J].Inflamm Res,2014,63(1):23-31.
[17]LI Y J,ZHOU J H,DU X X,et al.Dihydroartemisinin accentuates the anti-tumor effects of photodynamic therapy via inactivation of NF-κB in Eca109and Ec9706esophageal cancer cells[J].Cell Physiol Biochem,2014,33(5):1527-1536.
[18]ZHONG Z,SANCHEZ LOPEZ E,KARIN M.Autophagy,inflammation,and immunity:atroika governing cancer and its treatment[J].Cell,2016,166(2):288-298.
[19]ZOU K,LI Z,ZHANG Y,et al.Advances in the study of berberine and its derivatives:a focus on anti-inflammatory and anti-tumor effects in the digestive system[J].Acta Pharmacol Sin,2017,38(2):157-167.
[20]SAEKI K,WATANABE M,TSUBOI M,et al.Anti-tumour effect of metformin in canine mammary gland tumour cells[J].Vet J,2015,205(2):297-304.