枳术丸水煎液及其拆方含药血清对大鼠结肠Cajal间质细胞增殖和凋亡的影响

Efficacy of Zhizhuwan Decoction and Its Ingredient-contained Serums on Proliferation and Apoptosis of Rat Colon Interstitial Cells of Cajal

目的:探讨枳术丸水煎液及其拆方对大鼠结肠Cajal间质细胞(ICC)增殖和凋亡的影响及其分子机制,并从细胞分子层面分析中医攻补兼施法治疗慢传输型便秘可能的机制。方法:将40只大鼠随机分成白术组、枳实组、枳术丸组、空白血清组,每组10只。白术组灌以17.7 g·kg^-1·d^-1白术水煎液,枳实组灌以8.9 g·kg^-1·d^-1枳实水煎液,枳术丸组灌以26.4 g·kg^-1·d^-1枳术丸水煎液,空白血清组灌以无菌蒸馏水3 mL,每日1次,连续7 d,取血制备含药血清和空白血清。将4组含药血清分别配成5%,10%,15%,20%4个体积分数,干预24 h,观察ICC的数量、形态,用细胞增殖毒性检测试剂盒(CCK-8)检测各组细胞增殖情况,测定各组最佳干预体积分数。将大鼠结肠ICC细胞分为正常组、空白血清组、白术组、枳实组、枳术丸组,5-乙炔基-2'脱氧尿嘧啶核苷(EdU)检测各组ICC的增殖情况,流式细胞仪检测各组ICC的凋亡情况;蛋白免疫印迹法(Western blot)检测各组ICC中X连锁凋亡抑制蛋白(XIAP),增殖细胞核抗原(PCNA)蛋白表达情况。结果:与正常组比较,空白血清组、白术组、枳实组最佳干预体积分数均为10%,枳术丸组最佳干预体积分数为5%。与正常组比较,所有实验中空白血清组对ICC增殖和凋亡的影响没有明显差异;与正常组及空白血清组比较,白术组、枳实组、枳术丸组ICC增殖率有显著差异(P<0.05,P<0.01),与白术组和枳实组比较,枳术丸组对ICC增殖率明显增加(P<0.05,P<0.01);与枳实组比较,白术组对ICC增殖率变化不明显。与正常组及空白血清组比较,白术组、枳实组、枳术丸组ICC的凋亡率无明显差异;与正常组及空白血清组比较,白术组、枳实组、枳术丸组内的XIAP,PCNA蛋白水平均明显上调(P<0.05,P<0.01),三组间比较差异不明显。结论:枳术丸、枳实、白术在一定浓度下均能通过增加XIAP,PCNA蛋白表达促进ICC的增殖,且对ICC的凋亡无明显影响;以攻补兼施法立法的方剂枳术丸能促进ICC的增殖,并且其增殖效果优于攻伐单药枳实和补益单药白术。

Objective:To explore the efficacy and molecular mechanism of Zhizhuwan decoction and its ingredient-contained serums on the proliferation and apoptosis of rat colon interstitial cells of cajal(ICC),and make a molecule-level analysis of the possible mechanism of traditional Chinese medicine(TCM)purgation-tonifying therapy in treating slow transit constipation(STC).Method:A total of 40 rats were divided into Atractylodis Macrocephalae Rhizoma(AMR)group,Aurantii Fructus Immaturus(AFI)group,Zhizhuwan group and blank serum group on random basis,with 10 in each group.Baizhu group was given 17.7 g·kg^-1·d^-1 of AMR decoction by gavage,AFI group was given 8.9 g·kg^-1·d^-1 AFI decoction by gavage,Zhizhuwan group was given 26.4 g·kg^-1·d^-1 Zhizhuwan decoction by gavage,and blank serum group was given 3 mL sterile distilled water for 7 consecutive days,once a day.Drug-contained serums and blank serum were collected from blood of the above groups and diluted to 5%,10%,15%and 20%concentrations.Each concentration was intervened for 24 h and 48 h,and the amount and status of ICC were observed.The best intervening concentration and time for each group with cell counting kit-8(CCK-8)were determined.Rat colon ICC was divided into blank control group,blank serum group,AMR group,AFI group and Zhizhuwan group.ICC proliferation for each group was detected with EdU,ICC apoptosis for each group was detected by flow cytometry,and expressions of X-linked inhibitor of apoptosis protein(XIAP)and proliferating cell nuclear antigen(PCNA)were detected by Western blot.Result:Compared with the normal group,the best intervention concentration for blank serum group,AMR group and AFI group was 10%,while that for Zhizhuwan group was 5%.The best intervention times for the above groups were all 24 h.No distinct difference between the effect of blank control group and blank serum group on the proliferation and apoptosis of ICC was observed.In comparison with blank control group and blank serum group,AMR group,AFI group and Zhizhuwan group showed significant changes in ICC proliferation rate(P<0.05,P<0.01).There was a greater increase in ICC proliferation rate of Zhizhuwan group than that of AMR group and Zhizhu group(P<0.05,P<0.01),with no distinct difference between the changes of ICC proliferation rates in AMR group and AFI group.There was no significant difference between the changes of ICC apoptosis rates in AMR group,AFI group and Zhizhuwan group than in blank control group and blank serum group.There were significant increases in the expressions of XIAP and PCNA in AMR group,AFI group and Zhizhuwan group than in blank control group and blank serum group(P<0.05,P<0.01),but with little difference among the three groups.Conclusion:At certain concentrations,Zhizhuwan,AFI and AMR all have the effect in improving ICC proliferation by increasing XIAP and PCNA expressions,with no evident effect on the apoptosis of ICC,based on TCM purgation-tonifying therapy,Zhizhuwan has the effect in improving ICC proliferation,with a better effect than single administration with AFI or AMR.