制首乌含药血清对人乳腺癌T-47D细胞增殖及ER表达的影响

Effects of Processed Polygonum multiflorum Containing Serum on the Proliferation and the Expression of ER of Human Breast Cancer T-47D Cells

目的:研究制首乌含药血清对人乳腺癌T-47D细胞增殖及雌激素受体(ER)表达的影响,探讨其植物雌激素(PE)样作用。方法:将性未成熟SD大鼠随机分为空白组,戊酸雌二醇(Ev)组(阳性对照,0.12 mg/kg),制首乌低、高剂量组(0.75、3 g/kg,以生药量计)以及制首乌低、高剂量+Ev联用组(剂量同各药单用组),每组10只。空白组大鼠灌胃等体积水,各给药组大鼠灌胃相应药物,早晚各1次,连续4 d。末次给药2 h后采血,制备空白血清和含药血清。将T-47D细胞随机分为空白组,Ev组,制首乌低、高剂量组以及制首乌低、高剂量+Ev联用组,分置于含20%空白或相应含药血清的培养基中培养,采用CCK-8法检测各组细胞的增殖率(PR),采用Western blotting法和逆转录-聚合酶链反应法检测ER-α、ER-β蛋白及其mRNA的表达情况。结果:与空白组比较,各给药组细胞的PR[各给药组(24 h),除制首乌高剂量+Ev联用组外的其余各给药组(48、72 h)]均显著升高;且制首乌高剂量组(72 h)显著高于Ev组,制首乌低剂量组+Ev联用组(72 h)显著高于同剂量制首乌单用组,而制首乌高剂量+Ev联用组(72 h)显著低于同剂量制首乌单用组(P<0.05或P<0.01)。Ev组、制首乌高剂量组和制首乌低剂量+Ev联用组细胞中ER-α蛋白,各给药组细胞中ER-αmRNA和ER-β蛋白以及Ev组、制首乌低剂量组和制首乌+Ev联用组细胞中ER-βmRNA的相对表达量均显著升高;Ev组细胞中ER-α蛋白及其mRNA的相对表达量均显著高于制首乌单用和联用组;Ev组细胞中ER-β蛋白及其mRNA的相对表达量均显著低于制首乌低剂量+Ev联用组,但ER-βmRNA的相对表达量显著高于制首乌单用组和制首乌高剂量+Ev联用组;制首乌低剂量+Ev联用组细胞中ER-α、ER-β蛋白及其mRNA以及制首乌高剂量+Ev联用组细胞中ER-βmRNA的相对表达量均显著高于同剂量制首乌单用组,而制首乌高剂量+Ev联用组细胞中ER-α蛋白及其mRNA相对表达量均显著低于同剂量制首乌单用组(P<0.05或P<0.01)。结论:制首乌含药血清可促进人乳腺癌T-47D细胞的增殖,并可通过促进ER-α和ER-β蛋白及其mRNA的表达来发挥PE样作用。但上述作用弱于雌激素,且两者联合可能会拮抗雌激素的作用。

OBJECTIVE:To study the effects of processed Polygonum multiflorum containing serum on the proliferation and the expression of estrogen receptor(ER)of human breast cancer T-47D cells,and to investigate its phytoestrogen(PE)-like effect.METHODS:Sexually immature SD rats were randomly divided into estradiol valerate(Ev)group(positive control,0.12 mg/kg),processed P.multiflorum low-dose and high-dose groups(0.75,3 g/kg,by crude drug),low-dose and high-dose processed P.multiflorum+Ev groups(same dose as single drug group),with 10 rats in each group.Blank group was given constant volume of water intragastrically,and administration groups were given relevant medicine intragastrically;once day and night,for consecutive 4 days.Two hours after last administration,blank serum and containing serum were prepared.T-47D cells were also randomly divided into blank group,Ev group,low-dose and high-dose processed P.multiflorum groups,low-dose and high-dose processed P.multiflorum+Ev groups,and then were cultured in medium which contained 20%blank serum or drug containing serum.CCK-8 assay was used to detect proliferation rate(PR).Western blotting assay and RT-PCR were used to detect the protein and mRNA expression of ER-αand ER-β.RESULTS:Compared with blank group,PR of administration groups[each administration group(24 h),other administration groups(48,72 h)except for high-dose processed P.multiflorum+Ev group]were increased significantly;high-dose processed P.multiflorum group(72 h)was significantly higher than Ev group,and low-dose processed P.multiflorum+Ev group(72 h)was significantly higher than the same-dose processed P.multiflorum group;high-dose processed P.multiflorum+Ev group(72 h)was significantly lower than the same-dose processed P.multiflorum group(P<0.05 or P<0.01).Relative protein expression of ER-αin Ev group,high-dose processed P.multiflorum group and low-dose processed P.multiflorum+Ev group,relative mRNA expression of ER-αand protein expression of ER-β in administration groups,relative mRNA expression of ER-β in Ev group,low-dose processed P.multiflorum group and processed P.multiflorum+Ev groups were all increased significantly.Relative protein and mRNA expression of ER-αin Ev group were significantly higher than processed P.multiflorum groups and combination groups.Relative protein and mRNA expression of ER-β in Ev group were significantly lower than low-dose processed P.multiflorum+Ev group,but relative mRNA expression of ER-β was significantly higher than processed P.multiflorum groups and high-dose processed P.multiflorum+Ev group.Relative protein and mRNA expression of ER-αand ER-β in low-dose processed P.multiflorum+Ev group as well as relative mRNA expression of ER-β in high-dose processed P.multiflorum+Ev group were significantly higher than the same-dose processed P.multiflorum group.Relative protein and mRNA expression of ER-αin high-dose processed P.multiflorum+Ev group were significantly lower than the same-dose processed P.multiflorum group(P<0.05 or P<0.01).CONCLUSIONS:The processed P.multiflorum containing serum can promote the proliferation of human breast cancer T-47D cells,and play the PE-like role through promoting protein and mRNA expression of ER-αand ER-β.However,the above effects are weaker than estrogen,and the combination of the two may antagonize the effect of estrogen.