加味葛根芩连汤对湿热泄泻仔猪肠道炎症和损伤修复的作用

doi:10.11843/j.issn.0366-6964.2021.026

摘要/Abstract

摘要： 通过用加味葛根芩连汤治疗仔猪的湿热泄泻，探讨其对湿热泄泻仔猪肠道的修复作用。选取8~15日龄未断奶仔猪（大白×长白）30头，其中健康仔猪6头，为对照组；有粪色黄褐而臭、小便短赤、舌质红等症状的泄泻仔猪24头，分为湿热泄泻组、加味葛根芩连汤低剂量组、加味葛根芩连汤中剂量组和加味葛根芩连汤高剂量组，每组6头。对照组和湿热泄泻组仔猪灌服生理盐水，药物组仔猪灌服加味葛根芩连汤（按生药量，低、中、高剂量组药物添加量分别为2.5、5.0和10.0 g·d^-1），连续5 d。在服药的第0、3、5天记录各组仔猪的粪便评分。取对照组、湿热泄泻组、加味葛根芩连汤中剂量组仔猪十二指肠、空肠、回肠和结肠进行组织切片和H.E.染色，取十二指肠进行增殖性细胞核抗原（PCNA）免疫组化染色；用荧光定量PCR检测对照组、湿热泄泻组、加味葛根芩连汤中剂量组仔猪空肠TLR4、TNF-α、IL-1β和IL-6 mRNA表达量变化。湿热泄泻严重破坏了仔猪的十二指肠、空肠、回肠和结肠结构，尤其是十二指肠和空肠，十二指肠隐窝处PCNA表达量显著升高（P<0.05）。与对照组相比，湿热泄泻组仔猪空肠TLR4（P<0.05）、TNF-α（P<0.01）、IL-1β（P<0.01）和IL-6（P<0.05）mRNA表达量显著升高。相比于湿热泄泻组，加味葛根芩连汤显著降低了仔猪的粪便评分（P<0.05）。中剂量加味葛根芩连汤组仔猪的小肠和结肠的组织结构得到显著改善，十二指肠的肠腔中有不断脱落的绒毛团，绒毛中的PCNA表达量极显著升高（P<0.01）。与湿热泄泻组相比，加味葛根芩连汤中剂量组空肠TLR4（P<0.05）、TNF-α（P<0.01）、IL-1β（P<0.01）和IL-6（P<0.05）mRNA表达量显著降低。综上表明，加味葛根芩连汤可通过促进隐窝干细胞的增殖和降低炎症反应对湿热泄泻造成的仔猪肠道损伤进行修复。

关键词: 加味葛根芩连汤, 湿热泄泻, 仔猪, 肠道炎症, 损伤修复

Abstract: The purpose of the study was to research the effect of Jiawei Gegen Qinlian Decoction (JGQLD) on the regulation of intestine damage and repair in the piglets of damp-heat diarrhea (DHD). Six piglets (8-15 days old) were selected as control group. Twenty-four piglets (8-15 days old) that with the symptom of DHD were divided into 4 groups, including DHD group, low dose, middle dose and high dose of JGQLD group, 6 piglets per group. Piglets in the control group and the DHD group were given normal saline. Piglets in medicine groups were given the JGQLD for 5 consecutive days (the doses were recorded as dried medicinal herb, 2.5 g·d^-1 in the low dose group, 5.0 g·d^-1 in the middle dose, and 10.0 g·d^-1 in the high dose group). The fecal scores of piglets in all groups were recorded at 0, 3 and 5 day. In control group, DHD group and middle dose group of JGQLD, the duodenum, jejunum, ileum and colon of piglets were taken for tissue section and H.E. staining, while the duodenum was selected for proliferating cell nuclear antigen (PCNA) immune-histochemical staining. The mRNA expressions of TLR4, TNF-α, IL-1β and IL-6 in the jejunum of piglets from control group, DHD group and middle dose of JGQLD group were detected by real time PCR. In the DHD group, DHD destroyed the structure of small intestine and colon, especially the duodenum and jejunum. Compared with the control group, the PCNA expression in the duodenum crypt of the DHD group was significantly increased (P<0.05), the mRNA expressions of TLR4 (P<0.05), TNF-α (P<0.01), IL-1β (P<0.01) and IL-6 (P<0.05) in the jejunum of the DHD group were all increased significantly. Compared with the DHD group, the low, middle and high dose of JGQLD significantly decreased the fecal scores of piglets (P<0.05). The structures of duodenum, jejunum, ileum and colon in the middle dose of JGQLD group were improved and there were a few shedding villus in the lumen of duodenum. Compare with the DHD group, the PCNA expression in the duodenum villus of the middle dose group was extremely significantly increased (P<0.01), the mRNA expressions of TLR4 (P<0.05), TNF-α (P<0.01), IL-1β (P<0.01) and IL-6 (P<0.05) in the jejunum of the middle dose group were significantly decreased. JGQLD repaired the intestine damage of piglets with DHD by promoting the proliferation of crypt stem cells and reducing the intestinal inflammatory response.

Key words: Jiawei Gegen Qinlian Decoction, damp-heat diarrhea, piglets, intestinal inflammation, damage and repair

中图分类号:

S853.9

刘晓曦, 马云飞, 李焕荣, 刘凤华, 鲍明隆, 张继东, 林红英, 许剑琴. 加味葛根芩连汤对湿热泄泻仔猪肠道炎症和损伤修复的作用[J]. 畜牧兽医学报, 2021, 52(1): 246-255.

LIU Xiaoxi, MA Yunfei, LI Huanrong, LIU Fenghua, BAO Minglong, ZHANG Jidong, LIN Hongying, XU Jianqin. Effect of Jiawei Gegen Qinlian Decoction on the Regulation of Intestine Damage and Repair in the Piglets of Damp-heat Diarrhea[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 246-255.

参考文献 26

[1]	曹瑞丹,刘晓曦,李焕荣,等.关于北方部分地区猪场腹泻情况的调查报告[C]//中国猪业科技大会暨中国畜牧兽医学会2015年学术年会论文集.厦门,2015:286.CAO R D,LIU X X,LI H R,et al.Report on the diarrhea of pigs in part of northern China[C]//China pig science and Technology Conference and 2015 academic annual meeting of China animal husbandry and Veterinary Society.Xiamen,2015:286.(in Chinese)
[2]	CASBURN-JONES A C,FARTHING M J G.Management of infectious diarrhoea[J].Gut,2004,53(2):296-305.
[3]	冯春霞.Hp相关性胃炎脾胃湿热证本质初步研究[D].广州:广州中医药大学,2000.FENG C X.Basic studies on the essence of spleen-stomach damp-heat syndrome of Hp associated gastritis[D]. Guangzhou:Guangzhou University of Chinese Medicine,2000.(in Chinese)
[4]	高展翔.蒿芩清胆汤对流感病毒感染湿热证小鼠水通道蛋白影响的实验研究[D].广州:广州中医药大学,2012.GAO Z X.Effects of Haoqingqindan Decoction on aquaporin in damp-heat syndrome of mouse by influenza virus infection[D].Guangzhou:Guangzhou University of Chinese Medicine,2012.(in Chinese)
[5]	邢玉娟,杨帆,刘益,等.仔猪腹泻的辩证施治[J].中兽医学杂志,2017(6):15-17.XING Y J,YANG F,LIU Y,et al.Treatment of piglets diarrhea based on syndrome differentiation[J].Chinese Journal of Traditional Veterinary Science,2017(6):15-17.(in Chinese)
[6]	罗成宇,李点,姚欣艳,等.熊继柏教授辨治泄泻经验[J].中华中医药杂志,2014,29(9):2850-2853.LUO C Y,LI D,YAO X Y,et al.Experience of professor XIONG Ji-bo in differentiation and treatment of diarrhea[J].China Journal of Traditional Chinese Medicine and Pharmacy,2014,29(9):2850-2853.(in Chinese)
[7]	姚万玲,文艳巧,华永丽,等.郁金散对大肠湿热证大鼠回肠、结肠黏膜SIgA和炎性因子的影响[J].畜牧兽医学报,2019,50(10):2147-2155.YAO W L,WEN Y Q,HUA Y L,et al.Effects of Yujin powder on SIgA and inflammatory cytokines in ileal and colonic mucosa of large intestine dampness-heat syndrome rat[J].Acta Veterinaria et Zootechnica Sinica,2019,50(10):2147-2155.(in Chinese)
[8]	马琪.基于代谢组学和网络药理学的白头翁汤治疗湿热泄泻的作用机制研究[D].兰州:甘肃农业大学,2018.MA Q.Treatment effects of Pulsatilla decoction in dampness-heat diarrhea by metabolomics and network pharmacology approach[D].Lanzhou:Gansu Agricultural University,2018.(in Chinese)
[9]	李文龙.秦香止泻方对湿热泄泻的临床疗效观察及作用机制研究[D].长沙:湖南中医药大学,2011.LI W L.Study on clinical curative effects and mechanism of damp-heat diarrhea with QinXiang Anti-diarrhea[D].Changsha:Hunan University of Chinese Medicine,2011.(in Chinese)
[10]	林春发,郝永峰,刘娟.术苦芩总多糖对湿热泄泻仔猪小肠杯状细胞数量以及MUC-2和ITF-3 mRNA转录的影响[J].畜牧兽医学报,2019,50(6):1301-1311.LIN C F,HAO Y F,LIU J.Effect of polysaccharides from ZhuKuQin on the number of intestinal goblet cell and the transcription of MUC-2 and ITF-3 mRNA in the small intestine of damp-heat diarrhea piglets[J].Acta Veterinaria et Zootechnica Sinica,2019,50(6):1301-1311.(in Chinese)
[11]	钟宝华,李红彬,杨艺华,等.葛根芩连汤治疗仔猪菌痢肠炎[J].云南农业,2010(5):15.ZHONG B H,LI H B,YANG Y H,et al.Treatment of diarrhea enteritis in piglets with Gegen Qinlian Decoction[J].Yunnan Agriculture,2010(5):15.(in Chinese)
[12]	滕健樣.葛根芩连汤方证研究[D].北京:北京中医药大学,2012.TENG J Y.Research on the syndrome and the prescription of Gegen Qinlian Decoction[D].Beijing:Beijing University of Chinese Medicine,2012.(in Chinese)
[13]	LIU C S,LIANG X,WEI X H,et al.Gegen Qinlian decoction treats diarrhea in piglets by modulating gut microbiota and short-chain fatty acids[J].Front Microbiol,2019,10:825.
[14]	LI R Y,CHEN Y Y,SHI M J,et al.Gegen Qinlian decoction alleviates experimental colitis via suppressing TLR4/NF-κB signaling and enhancing antioxidant effect[J].Phytomedicine,2016,23(10):1012-1020.
[15]	HU C H,SONG J,YOU Z T,et al.Zinc oxide-montmorillonite hybrid influences diarrhea,intestinal mucosal integrity,and digestive enzyme activity in weaned pigs[J].Biol Trace Elem Res,2012,149(2):190-196.
[16]	续畅,钟萌,马致洁,等.葛根芩连汤的现代研究进展[J].吉林中医药,2015,35(6):629-631,632.XU C,ZHONG M,MA Z J,et al.Research status of Gegen Qinlian decoction[J].Jilin Journal of Traditional Chinese Medicine,2015,35(6):629-631,632.(in Chinese)
[17]	余林中,伍杰勇,罗佳波,等.葛根芩连汤配伍抗腹泻药效实验研究[J].中成药,2005,27(3):300-304.YU L Z,WU J Y,LUO J B,et al.Experimental study on anti-diarrhae effect of Gegenqinlian Decoction and its compounds[J].Chinese Traditional Patent Medicine,2005,27(3):300-304.(in Chinese)
[18]	陈阳,崔波,范燕豪,等.葛根芩连汤中各成分在正常与大肠湿热证模型大鼠的肠吸收差异研究[J].中国中药杂志,2020,45(1):169-178.CHEN Y,CUI B,FAN Y H,et al.Differences in intestinal absorption of Gegen Qinlian Decoction between normal rats and rats with large intestinal damp-heat syndrome[J].China Journal of Chinese Materia Medica,2020,45(1):169-178.(in Chinese)
[19]	汤梦倩,刘学红.增殖性细胞核抗原和小肠发育的关系[J].中国组织工程研究,2012,16(18):3380-3382.TANG M Q,LIU X H.Relationship between proliferating cell nuclear antigen and intestinal development[J].Chinese Journal of Tissue Engineering Research,2012,16(18):3380-3382.(in Chinese)
[20]	FAN W,LIU Y L,WU Z F,et al.Effects of rosiglitazone,an agonist of the peroxisome proliferator-activated receptor γ,on intestinal damage induced by Escherichia coli lipopolysaccharide in weaned pigs[J].Am J Vet Res,2010,71(11):1331-1338.
[21]	LIU X X,LIU F H,MA Y F,et al.Effect of puerarin,baicalin and berberine hydrochloride on the regulation of IPEC-J2 cells infected with enterotoxigenic Escherichia coli[J].Evid Based Complement Alternat Med,2019,2019:7438593.
[22]	CHERNUY C,HONVO-HOUETO E,BRUNEAU A,et al.Microbiota matures colonic epithelium through a coordinated induction of cell cycle-related proteins in gnotobiotic rat[J].Am J Physiol Gastrointest Liver Physiol,2010,299(2):G348-G357.
[23]	刘晓曦,董杰,李敏霞,等.黄芩水提液对肠炎模型小鼠空肠损伤修复的作用[J].畜牧兽医学报,2020,51(2):392-398.LIU X X,DONG J,LI M X,et al.Effect of the Scutellaria baicalensis water extraction on the regulation of jejunum damage and repair in the model of enteritis mice[J].Acta Veterinaria et Zootechnica Sinica,2020,51(2):392-398.(in Chinese)
[24]	WULLAERT A.Role of NF-κB activation in intestinal immune homeostasis[J].Int J Med Microbiol,2010,300(1):49-56.
[25]	LUO L,LIU Y Q,CAI X,et al.Bletilla striata polysaccharides ameliorates lipopolysaccharide-induced injury in intestinal epithelial cells[J].Saudi J Gastroenterol,2019,25(5):302-308.
[26]	CAPALDO C T,NUSRAT A.Cytokine regulation of tight junctions[J].Biochim Biophys Acta,2009,1788(4):864-871.