枳术丸汤剂含药血清对大鼠结肠Cajal间质细胞增殖及PLC-γ信号通路的影响

Effect of Serum Containing Zhizhuwan Decoction(枳术丸汤剂)on Proliferation and Colonic Interstitial Cells Cajal and PLC-γSignaling Pathway in Rats

目的探讨枳术丸治疗慢传输型便秘的可能作用机制。方法20只SD雄性大鼠随机分为枳术丸汤剂组、空白组,每组10只。枳术丸汤剂组大鼠每日给予浓度为2 g/ml枳术丸汤剂5.8 g灌胃,连续7天,空白组正常饲养。末次给药后1 h腹主动脉采血离心制备含药血清。采用CCK8法观察磷脂酶C-γ(PLC-γ)特异性抑制剂U73122对大鼠结肠原代Cajal间质细胞的细胞抑制率来筛选药物浓度,观察5%、10%、15%、20%浓度的枳术丸汤剂含药血清对Cajal间质细胞的细胞增殖率筛选药物浓度,并分别培养12、24、48 h来筛选用药时间。取二或三代Cajal间质细胞接种至96孔板中,每孔1×10^(4)个细胞,每组重复3孔。实验分为正常组、模型组(正常细胞+筛选浓度的U73122)、空白血清组(模型组+10%空白血清)、枳术丸汤剂血清组(模型组+筛选浓度枳术丸含药血清)。干预时间为实验筛选出的时间。免疫组化法检测磷脂酶C-γ1(PLC-γ1)、磷脂酶C-γ2(PLC-γ2)阳性表达,Western blot法检测PLC-γ1、PLC-γ2蛋白表达,RT-PCR法检测PLC-γ1、PLC-γ2基因表达。结果筛选出U73122最佳实验药物浓度为30μmol/L,枳术丸汤剂组最佳用药浓度为5%,实验最佳干预时间为24 h。与正常组比较,模型组PLC-γ1、PLC-γ2平均光密度值、蛋白及基因表达均降低(P<0.01);与模型组比较,枳术丸汤剂血清组、空白血清组PLC-γ1、PLC-γ2平均光密度值、蛋白及基因表达均升高(P<0.05或P<0.01);与空白血清组比较,枳术丸汤剂血清组PLC-γ1、PLC-γ2阳性表达、蛋白及基因表达明显增多(P<0.01)。结论枳术丸汤剂可能通过激活Cajal间质细胞PLC-γ信号通路,调控细胞增殖,从而促进胃肠运动。

Objective To explore the possible mechanism of Zhizhuwan Decoction(枳术丸汤剂,ZD)for treatment of slow transit constipation(STC).Methods Twenty SD male rats were randomized into ZD group and blank group,with 10 rats in each group.The rats in the ZD group were given 5.8 g ZD by gavage at a concentration of 2 g/ml per day for seven consecutive days,while those in the blank group were fed normally.One hour after the last administration,blood was collected from the abdominal aorta and centrifuged to prepare the medicated serum.CCK8 method was used to assess the cell inhibition rate of U73122,a specific inhibitor of phospholipase C-γ(PLC-γ),on primary interstitial cells of Cajal(ICC);the concentration of medicated serum was selected from 5%,10%,15%and 20%by observing the effect on cell proliferation of ICC;the medication time was selected from 12 h,24 h and 48 h based on the results.Second or third generation of ICC were seeded into a 96-well plate,with 1×10^(4) cells per well and three repeated wells for each of the three groups,which were normal group,model group(normal cells plus U73122 at the selected concentration),blank serum group(model group plus 10%blank serum)and ZD-containing serum group(model group plus ZD-containing serum at the selected concentration);the administrations lasted for the selected time duration.The positive expression of PLC-γ1 and PLC-γ2 were detected by immunohistochemistry,and the protein and gene expression of PLC-γ1 and PLC-γ2 were detected through Western blotting and RT-PCR,respectively.Results The selected best concentration was 30μmol/L of U73122,and was 5%of ZD;and the selected administration time was 24 h.Compared to the normal group,the model group had decreased PLC-γ1,PLC-γ2 average optical density,protein and gene expression(P<0.01);compared to the model group,the ZD-containing serum group and blank serum group had higher PLC-γ1 and PLC-γ2 average optical density,protein and gene expression(P<0.05 or P<0.01);compared to the blank serum group,the ZD-containing serum group had significantly increased positive expression of PLC-γ1,PLC-γ2,as well as protein and gene expression(P<0.01).Conclusion ZD may regulate cell proliferation through activation of the PLC-γsignaling pathway of ICC,so as to promote the gastrointestinal motility.