脾虚四号方含药血清对功能性腹泻脾虚证大鼠离体结肠平滑肌细胞的影响

摘要

目的 从细胞水平角度,观察脾虚四号方含药血清干预离体培养功能性腹泻脾虚证模型大鼠的结肠平滑肌细胞的变化.方法 对功能性腹泻脾虚证模型大鼠进行提取离体结肠平滑肌细胞并原代培养,将其分为6组:空白对照组、模型组、脾虚四号方低、中、高剂量组、蒙脱石散组,各组分别5%含药血清干预48 h,用细胞增殖和毒性检测(CCK-8)法测定细胞增殖活性,实时荧光定量PCR检测各组相关脑肠肽的基因表达.结果 CCK-8法检测结果显示:与空白对照组相比,模型组结肠平滑肌细胞OD值极显著降低(P<0.01);与模型组相比,各含药血清干预组结肠平滑肌细胞OD值极显著升高(P<0.01).实时荧光定量PCR检测结果显示:与空白对照组相比,模型组胆囊收缩素(CCK)、血管活性肠肽(VIP)、生长抑素(SS) mRNA表达均显著降低(P<0.05).与模型组相比,各含药血清干预组均有表达升高趋势,脾虚四号方低剂量组较模型组CCK mRNA表达显著升高(P<0.05),脾虚四号方低、中、高剂量组较模型组VIP mRNA表达极显著升高(P<0.01).结论 脾虚四号方含药血清能够有效促进模型结肠平滑肌细胞的增殖活性,脑肠肽CCK、VIP、SS mRNA表达变化可能有利于进一步阐释功能性腹泻脾虚证在脑肠肽胃肠激素方面发病机制.%Objective To study the cellular changes of colonic smooth muscle cells in rats with spleen-deficiency functional diarrhea treated with Pixu Sihao Fang (PXSHF, No.4 Formula for Spleen Deficiency) dosed serum.Methods Colonic smooth muscle cells from functional diarrhea spleen-deficiency rats were extracted for primary culture and then divided into the following six groups: normal control group, model group, PXSHF low-dose, medium-dose, and high-dose groups, and montmorillonite powder (Mnt) group.All the dosed serum groups were treated with 5% drug serum for 48 h.CCK-8 test for detecting the cell proliferation activity and QRT-PCR test for checking the expression of brain-gut petide mRNA were performed.Results OD value of the model group was significantly lower than that of the control group (P<0.01) in CCK-8 test while the OD value of the drug serum groups were significantly higher than that of the model group (P<0.01).QRT-PCR test showed that the CCK/VIP/SS mRNA expression of the model group was significant lower than that of the control group (P<0.05).Compared with the model group, the CCK/VIP/SS mRNA expression in each drug serum group presented with an increase tendency.CCK mRNA expression of the group treated with low-dose PXSHF dosed serum was higher than of the model group (P<0.05).VIP mRNA expression of the groups treated with PXSHF dosed serum were significantly higher than that of the model group (P<0.01).Conclusion PXSHF dosed serum can effectively promote the proliferation activity of the colonic smooth muscle cells.Changes in the expression of CCK, VIP and SS mRNA might be indicative of the role of brain-gut peptide in the development of functional diarrhea with spleen deficiency pattern.