枳术丸对脾虚证慢传输型便秘小鼠肠道运动及PLC-γ_1/PLC-γ_2信号通路的影响
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摘要
目的:探讨枳术丸对脾虚证慢传输型便秘(STC)小鼠肠道传导功能的干预作用,及对小鼠结肠组织磷脂酶C-γ_1(PLC-γ_1)/磷脂酶C-γ_2(PLC-γ_2)信号通路表达的影响。方法:将无特定病原体(SPF)健康小鼠随机分为正常组与造模组。采用番泻叶灌胃造成脾虚状态,然后控制饮食饮水的方法造成脾虚便秘的小鼠模型,造模完成后将模型组小鼠随机分为模型组、枳术丸组、莫沙必利组,枳术丸组按9. 0 g·kg~(-1)·d~(-1)给药,莫沙必利组按2. 5 mg·kg~(-1)·d~(-1)给药,模型组和正常组给予等剂量的蒸馏水,连续给药7 d,治疗完成后用印度墨水在结肠中推进的长度计算小鼠肠道推进率;用D-木糖试剂盒测定小鼠血清D-木糖含量;用苏木素-伊红(HE)染色观察小鼠结肠组织病理改变;采用免疫组化法及蛋白免疫印迹法(Western blot)检测小鼠结肠组织PLC-γ_1,PLC-γ_2蛋白的表达水平;采用实时荧光定量PCR(Real-time PCR)检测PLC-γ_1,PLC-γ_2mRNA表达水平。结果:与正常组比较,模型组小鼠肠道推进率及血清D-木糖值均显著降低(P <0. 01),HE染色观察结肠黏膜明显充血、水肿,结肠组织PLC-γ_1,PLC-γ_2蛋白与mRNA表达水平均显著减少(P <0. 01);与模型组比较,枳术丸组和莫沙必利组小鼠肠道推进率及血清D-木糖含量均显著增加(P <0. 01),HE染色观察结肠黏膜充血、水肿及炎症细胞浸润改善,PLC-γ_1,PLC-γ_2蛋白与mRNA表达水平均显著升高(P <0. 01)。结论:枳术丸可有效促进脾虚便秘小鼠的肠道运动,改善便秘症状,其相关的作用机制可能与提高脾虚便秘小鼠结肠组织中PLC-γ_1与PLC-γ_2的表达有关。
Objective: To explore the effect of Zhizhuwan on intestinal conduction and expressions of Phospholipase C-γ_1( PLC-γ_1)/Phospholipase C-γ_2( PLC-γ_2) signaling pathway of slow transit constipation( STC) with spleen deficiency syndrome. Method: Special pathogen free( SPF) healthy mice were randomly divided into normal group and model making group. Folium Sennae gavage was used to induce the spleen deficiency status,and then diet and drinking water were controlled to establish the mice model of spleen deficiency constipation. After the modeling,the mice in modelling group were randomly divided into model group,Zhizhuwan group and mosapride group. Zhizhuwan group was given drug at the dose of 9. 0 g·kg~(-1)·d~(-1),mosapride group was given 2. 5 mg·kg~(-1)·d~(-1),model group and normal group were given the equal dose of distilled water for 7 consecutive days. At the end of the treatment,the length of Indian ink in the colon was used to calculate the intestinal propulsion rate of the mice. The D-xylose kit was used to determine the content of D-xylose in serum of mice. The hematoxylin eosin( HE) staining was used to observe the pathological changes of colon tissues in mice.The immunohistochemistry and Western blot were used to detect the expression levels of PLC-γ_1 and PLC-γ_2 proteins in colon tissues of mice. Real-time PCR was used to detect the mRNA expression levels of PLC-γ_1 and PLC-γ_2. Result: Compared with normal group,the intestinal propulsive rate and the serum D-xylose value in model group were significantly decreased( P < 0. 01). Obvious hyperemia and edema of colonic mucosa were observed by HE staining. The protein and mRNA expression levels of PLC-γ_1 and PLC-γ_2 in colon tissues were significantly decreased( P < 0. 01). Compared with model group,the intestinal propulsion rate and the content of D-xylose in Zhizhuwan group and mosapride group were significantly increased( P < 0. 01). Colonic mucosal hyperemia,edema and inflammatory cell infiltration were improved according to HE staining. The protein and mRNA expression levels of PLC-γ_1 and PLC-γ_2 were significantly increased( P < 0. 01). Conclusion: Zhizhuwan can promote the intestinal movement in slow transit constipation model mouse with spleen deficiency syndrome,and alleviate the symptoms of constipation in mice. The related mechanism may be related to the increase of the expressions of PLC-γ_1 and PLC-γ_2 in colon tissues of mice with spleen deficiency and constipation.
Objective: To explore the effect of Zhizhuwan on intestinal conduction and expressions of Phospholipase C-γ_1( PLC-γ_1)/Phospholipase C-γ_2( PLC-γ_2) signaling pathway of slow transit constipation( STC) with spleen deficiency syndrome. Method: Special pathogen free( SPF) healthy mice were randomly divided into normal group and model making group. Folium Sennae gavage was used to induce the spleen deficiency status,and then diet and drinking water were controlled to establish the mice model of spleen deficiency constipation. After the modeling,the mice in modelling group were randomly divided into model group,Zhizhuwan group and mosapride group. Zhizhuwan group was given drug at the dose of 9. 0 g·kg~(-1)·d~(-1),mosapride group was given 2. 5 mg·kg~(-1)·d~(-1),model group and normal group were given the equal dose of distilled water for 7 consecutive days. At the end of the treatment,the length of Indian ink in the colon was used to calculate the intestinal propulsion rate of the mice. The D-xylose kit was used to determine the content of D-xylose in serum of mice. The hematoxylin eosin( HE) staining was used to observe the pathological changes of colon tissues in mice.The immunohistochemistry and Western blot were used to detect the expression levels of PLC-γ_1 and PLC-γ_2 proteins in colon tissues of mice. Real-time PCR was used to detect the mRNA expression levels of PLC-γ_1 and PLC-γ_2. Result: Compared with normal group,the intestinal propulsive rate and the serum D-xylose value in model group were significantly decreased( P < 0. 01). Obvious hyperemia and edema of colonic mucosa were observed by HE staining. The protein and mRNA expression levels of PLC-γ_1 and PLC-γ_2 in colon tissues were significantly decreased( P < 0. 01). Compared with model group,the intestinal propulsion rate and the content of D-xylose in Zhizhuwan group and mosapride group were significantly increased( P < 0. 01). Colonic mucosal hyperemia,edema and inflammatory cell infiltration were improved according to HE staining. The protein and mRNA expression levels of PLC-γ_1 and PLC-γ_2 were significantly increased( P < 0. 01). Conclusion: Zhizhuwan can promote the intestinal movement in slow transit constipation model mouse with spleen deficiency syndrome,and alleviate the symptoms of constipation in mice. The related mechanism may be related to the increase of the expressions of PLC-γ_1 and PLC-γ_2 in colon tissues of mice with spleen deficiency and constipation.
引文
[1]魏东.慢传输型便秘外科治疗进展[J].世界华人消化杂志,2017,25(16):1438-1445.
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[7] Lee S,Gim H,Shim J H,et al. The traditional herbal medicine,Ge-Gen-Tang,inhibits pacemaker potentials by nitric oxide/c GMP dependent ATP-sensitive K+channels in cultured interstitial cells of Cajal from mouse small intestine[J]. J Ethnopharmacol,2015,170:201-209.
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[11] Dieni C A,Storey K B. Protein kinase C in the wood frog,Rana sylvatica:reassessing the tissue-specific regulation of PKC isozymes during freezing[J]. Peer J,2014,2(1):558.
[12]王芳,于欢,李风琴,等.枳实制白术的炮制工艺优选及HPLC特征图谱分析[J].中国实验方剂学杂志,2018,24(17):6-13.
[13]刘富林,易健,刘柏炎,等.超微枳术丸对功能性消化不良小鼠胃肠运动及MTL、GAS的影响[J].中国中医急症,2011,20(4):594-595,604.
[14]刘富林,谷井文,易健,等.枳术丸超微颗粒与传统汤剂治疗脾虚气滞型功能性消化不良的比较研究[J].湖南中医药大学学报,2010,30(5):52-54.
[15]夏旭婷,樊俊阳,王碧玉,等.脾虚证慢传输型便秘小鼠模型构建方法的比较研究[J].湖南中医药大学学报,2014,34(12):4-7,81.
[16]吴勉华,王新月.中医内科学[M].北京:中国中医药出版社,2012:237-238.
[17]余瀛鳌.枳术丸源流及其变方[J].中医杂志,1980(5):68.
[18] Rebecchi M, Pentyala S. Structure, function, and control of phosphoinositide-specific phospholipase C[J]. Physiol Rev,2000,80(4):1291-1335.
[19] Rhee S G,Choi K D,PAN G,et al. Regulation of inositol phospholipid-specific phospholipase C isozymes[J]. J Biol Chem,1992,267(18):12393-12396.
[20] Kassis J,Radinsky R,Wells A. Motility is rate-limiting for invasion of bladder carcinoma cell lines[J]. Int J Biochem Cell Biol,2002,34(7):762-775.
[21] Gresset A,Sondek J T, Harden K, et al. The phospholipase C isozymes and their regulation[J].Subcell Biochem,2012,58(1):61-94.
[22] Hashimoto A,Takeda K,Inaba M,et al. Cutting edge:essentialrole of phospholipase C-gamma 2 in B cell development and function[J]. J Immunol,2000,165:1738-1742.
[23]邹颖,郑学宝,戴世学,等.枳术汤对脾虚便秘小鼠Cajal间质细胞和一氧化氮表达的影响[J].时珍国医国药,2011,22(3):591-593.
[24]翁萍,王文凯,张晓婷.白术不同炮制品对脾虚小鼠胃肠功能的影响[J].江西中医药,2008,46(5):30-32.
[2] LIU M C,XIE M Z,MA B,et al. Effect of Da-ChengQi decoction on the repair of the injured enteric nerveinterstitial cells of Cajal-smooth muscle cells network in multiple organ dysfunction syndrome[J]. Evid-Based Compl Alt,2014,doi. org/10. 1155/2014/596723.
[3] Hall K A, Ward S M, Cobine C, et al. Spatial organization and coordination of slow waves in the mouse anorectum[J]. J Physiol,2014,592(17):3813-3829.
[4] ZHAO Y J,LI G X,SUN X M,et al. Changes of amount and ultrastructure of interstitial cells of Cajal in rats with severe acute pancreatitis and their importance[J]. Int J Clin Exp Med,2015,8(4):5492-5498.
[5] ZHANG M Y, LI S H, HUANG G L, et al.Identification of a novel microRNA signature associated with intrahepatic cholangiocarcinoma(ICC)patient prognosis[J]. BMC Cancer,2015,15(1):64.
[6] Shin D H,Lee M J,JIAO H Y,et al. Regulatory roles of endogenous mitogen-activated protein kinases and tyrosine kinases in the pacemaker activity of colonic interstitial cells of Cajal[J]. Pharmacology,2015,96(1/2):16-24.
[7] Lee S,Gim H,Shim J H,et al. The traditional herbal medicine,Ge-Gen-Tang,inhibits pacemaker potentials by nitric oxide/c GMP dependent ATP-sensitive K+channels in cultured interstitial cells of Cajal from mouse small intestine[J]. J Ethnopharmacol,2015,170:201-209.
[8] Angeli T R,CHENG L K,DU P,et al. Loss of interstitial cells of Cajal and patterns of gastric dysrhythmia in patients with chronic unexplained nausea and vomiting[J]. Gastroenterology,2015,149(1):56-66.
[9] Adachi Y,Ishii Y,Yoshimoto M,et al. Phenotypic alteration of interstitial cells of Cajal in idiopathic sigmoid megacolon[J]. J Gastroenterol,2008,43(8):626-631.
[10]夏旭婷.枳术丸对脾虚证慢传输型便秘小鼠结肠黏膜C-kit、SCF表达的影响[D].长沙:湖南中医药大学,2015.
[11] Dieni C A,Storey K B. Protein kinase C in the wood frog,Rana sylvatica:reassessing the tissue-specific regulation of PKC isozymes during freezing[J]. Peer J,2014,2(1):558.
[12]王芳,于欢,李风琴,等.枳实制白术的炮制工艺优选及HPLC特征图谱分析[J].中国实验方剂学杂志,2018,24(17):6-13.
[13]刘富林,易健,刘柏炎,等.超微枳术丸对功能性消化不良小鼠胃肠运动及MTL、GAS的影响[J].中国中医急症,2011,20(4):594-595,604.
[14]刘富林,谷井文,易健,等.枳术丸超微颗粒与传统汤剂治疗脾虚气滞型功能性消化不良的比较研究[J].湖南中医药大学学报,2010,30(5):52-54.
[15]夏旭婷,樊俊阳,王碧玉,等.脾虚证慢传输型便秘小鼠模型构建方法的比较研究[J].湖南中医药大学学报,2014,34(12):4-7,81.
[16]吴勉华,王新月.中医内科学[M].北京:中国中医药出版社,2012:237-238.
[17]余瀛鳌.枳术丸源流及其变方[J].中医杂志,1980(5):68.
[18] Rebecchi M, Pentyala S. Structure, function, and control of phosphoinositide-specific phospholipase C[J]. Physiol Rev,2000,80(4):1291-1335.
[19] Rhee S G,Choi K D,PAN G,et al. Regulation of inositol phospholipid-specific phospholipase C isozymes[J]. J Biol Chem,1992,267(18):12393-12396.
[20] Kassis J,Radinsky R,Wells A. Motility is rate-limiting for invasion of bladder carcinoma cell lines[J]. Int J Biochem Cell Biol,2002,34(7):762-775.
[21] Gresset A,Sondek J T, Harden K, et al. The phospholipase C isozymes and their regulation[J].Subcell Biochem,2012,58(1):61-94.
[22] Hashimoto A,Takeda K,Inaba M,et al. Cutting edge:essentialrole of phospholipase C-gamma 2 in B cell development and function[J]. J Immunol,2000,165:1738-1742.
[23]邹颖,郑学宝,戴世学,等.枳术汤对脾虚便秘小鼠Cajal间质细胞和一氧化氮表达的影响[J].时珍国医国药,2011,22(3):591-593.
[24]翁萍,王文凯,张晓婷.白术不同炮制品对脾虚小鼠胃肠功能的影响[J].江西中医药,2008,46(5):30-32.