【作者】 王婷；

【导师】 万军；

【作者基本信息】 西南交通大学 ， 药学， 2021， 硕士

【摘要】 目的:枳实是“麸炒入药”的代表药味,临床多用于治疗功能性消化不良（Functional dyspepsia,FD）等消化系统疾病,其生用刺激性较强,麸炒后可缓和燥性,提高疗效,本课题以柚皮苷、橙皮苷、新橙皮苷为评价指标,研究麸炒对枳实主要成分的影响;以FD大鼠为模型,研究麸炒前后枳实对于FD模型大鼠胃肠道激素,胃组织内质网应激相关因子,盲肠组织肠道菌群多样性的影响,以期初步阐明枳实治疗FD的作用机制,为麸炒类中药研究提供一定的参考和借鉴。方法:1.参照《中国药典》2015版制备枳实麸品,采用HPLC法检测柚皮苷、橙皮苷、新橙皮苷等总黄酮类成分麸炒前后含量变化。2.SPF级SD大鼠60只随机分为空白组、模型组、阳性组、枳实生品组、枳实麸品组,除空白组外,其余组采用“郭海军夹尾法”加以改进连续刺激21 d建立FD大鼠模型,通过一般状态、体重、小肠推进率、胃排空率及HE染色验证造模成功与否。造模结束后分别按空白组、模型组(氯化钠注射液10 m L·kg^-1·d^-1),阳性组(多潘立酮溶液2.7 mg·kg^-1·d^-1),枳实生品组(枳实生品溶液9 g·kg^-1·d^-1),枳实麸品组(枳实麸品溶液9 g·kg^-1·d^-1)灌胃给药14 d,ELISA法检测血清胃动素（MTL）、胃泌素（GAS）、生长抑素（SS）的含量。3.采用ELISA、RT-PCR、WB法检测大鼠胃组织葡萄糖调节蛋白（GRP78）、肌醇需求酶1（IRE1）、c-Jun氨基末端激酶（JNK）等内质网应激相关因子的表达,采用WB法检测胃组织中凋亡因子Bcl-2、Bax蛋白变化,探究枳实麸炒前后对FD大鼠的治疗作用是否与胃组织内质网应激有关。4.通过16S r DNA扩增子测序技术对大鼠盲肠粪便菌群进行测序,分别进行Alpha多样性、Beta多样性、不同分类水平下菌群的相对丰度及差异物种分析,探究FD大鼠肠道菌群结构变化以及枳实对FD大鼠肠道菌群结构的调节作用。结果:1.参照《中国药典》2015版一部,以麦麸:枳实投料比为10:1,中火炒至枳实表面色变深,筛去麦麸,放凉,得枳实麸品。确定含量测定的最佳色谱条件为:色谱柱:Alltima TMC₁₈柱（250 mm×4.6 mm,5μm）;流动相:乙腈-0.1%甲酸（20:80）;流速:1.0 m L/min;检测波长:284 nm;进样量:10μL;柱温:30℃,且麸炒后柚皮苷、橙皮苷及新橙皮苷等黄酮类成分含量均有所降低。2.连续造模21 d后,大鼠精神状态差,活跃度低,毛色枯黄脱落,体重、肠推进率及胃排空率明显降低（P<0.01）,HE染色发现模型组大鼠胃组织无明显病理学变化,表明FD大鼠模型建立成功。给药14 d后,与模型组相比,阳性组、麸品组大鼠体重明显增加（P<0.01）,GAS、MTL含量明显增加（P<0.01）,SS含量明显降低（P<0.01）。与模型组相比,生品组大鼠体重增加（P<0.05）,GAS、MTL分泌增加（P<0.05）,SS分泌降低（P<0.05）。表明枳实麸品可更好改善FD大鼠胃肠动力,增加胃肠激素分泌,进而改善FD大鼠一般状态。3.与空白组相比,模型组大鼠胃组织中IRE1、GRP78等内质网应激相关因子的m RNA及蛋白表达明显增加（P<0.05）,JNK在m RNA水平上表达显著增加（P<0.05）,与模型组相比,阳性组、麸品组大鼠胃组织中IRE1、GRP78m RNA及蛋白表达显著降低,JNK m RNA表达降低。与空白组相比,模型组大鼠胃组织Bcl-2蛋白表达降低,Bax蛋白表达增加（P<0.05）,与模型组相比,阳性组、麸品组大鼠胃组织Bcl-2蛋白表达增加,Bax蛋白表达降低（P<0.05）。4.采用16S r DNA扩增子测序技术对大鼠肠道菌群进行分析,结果发现,FD模型大鼠肠道菌群多样性及丰度下降,在门水平上,与空白组相比,厚壁菌门相对丰度降低,拟杆菌门、变形菌门相对丰度增加,结合LEfse分析结果及相关文献推测毛螺菌科、瘤球菌科、普雷沃氏菌科丰度变化可能与FD的发生密切相关,给药治疗后可调整菌群结构,且枳实麸品的作用优于生品。结论:枳实麸炒后以柚皮苷、橙皮苷及新橙皮苷为代表的黄酮类成分显著降低。药效学考察发现,枳实生品及麸品可增加FD大鼠胃排空率及小肠推进率,调整胃肠道激素分泌,改善一般状态,其作用机制可能与抑制胃组织IRE1、GRP78、JNK等内质网应激相关因子表达,减缓内质网应激现象,降低凋亡蛋白表达从而增强胃动力有关,且麸炒后效果更佳。肠道菌群中毛螺菌科、瘤球菌科、普雷沃氏菌科相对丰度的变化可能与FD有关,枳实麸品对FD大鼠肠道菌群结构的调节作用优于生品。综上所述,枳实麸炒后其黄酮类成分虽有所降低,但其仍可通过调节胃肠道激素分泌、抑制内质网应激及调节肠道菌群多样性及丰度缓解FD相关症状。更多还原

【Abstract】 Objective:As the representative medicine of"stir fried with bran",Fructus aurantii Immaturus（FAI）is often used in the treatment of digestive system diseases such as functional dyspepsia（FD）.Its raw product has strong irritation to human body but stir fried with bran can alleviate its side effects and improve efficacy.In this study,naringin,hesperidin and neohesperidin were used as evaluation indexes to study the effect of stir fried with bran on the main components of FAI.The effects of FAI and its stir fried with bran product on gastrointestinal hormones,stress related factors of endoplasmic reticulum and intestinal flora diversity of cecum were studied in FD rats.So as to clarify the mechanism of FAI in the treatment of FD and provide some reference for the research of bran fried medicine.Method:1.We prepared fried FAI with bran with reference to Chinese Pharmacopoeia（2015 edition）,the content of total flavonoids such as naringin,hesperidin,neohesperidin in FAI and its stir fried with bran product were determined by High Performance Liquid Chromatography（HPLC）.2.Sixty SPF SD rats were randomly divided into blank group（BG）,model group（MG）,positive group（PG）,raw Fructus aurantii Immaturus group（RFAI）and fried Fructus aurantii Immaturus with bran group（FFAIWB）.Except for BG,the rest groups were treated with the modified"Guo Haijun tail-clamping method"for 21 d to establish the FD rats model.The general state,body weight,gastric emptying rate,intestinal propulsion rate and HE staining were used to evaluated the model.After the model was built,all rats were given the corresponding solution by gavage for 14 days.The rats in BG,MG were given sodium chloride injection(10 m L·kg^-1·d^-1),the rats in PG were given domperidone solution(2.7 mg·kg^-1·d^-1),the rats in RFAI were given raw FAI decoction(9 g·kg^-1·d^-1),the rats in FFAIWB were given stir fried with bran FAI decoction(9 g·kg^-1·d^-1).After administration,the levels of serum motilin（MTL）,gastrin（GAS）and somatostatin（SS）were detected by Enzyme linked immunosorbent assay（ELISA）.3.The expression of glucose regulatory protein（GRP78）,inositol demand enzyme 1（IRE1）,c-jun-terminal kinase（JNK）were detected by ELISA,reverse transcription polymerase chain reaction（RT-PCR）and western blot（WB）respectively.The expression of Bcl-2 and Bax in gastric tissue were detected by WB.So we can study whether the regulating effect of FAI on FD rats is related to the gastric endoplasmic reticulum stress.4.The intestinal flora of FD rats was sequenced by 16S r DNA amplicon sequencing technology.Alpha diversity,Beta diversity,relative abundance of flora under different classification levels were analyzed respectively to explore the changes in intestinal flora structure of FD rats and the regulatory effect of FAI on intestinal flora structure of FD rats.Results:1.According to the Chinese Pharmacopoeia 2015 Volume I,the ratio of bran to FAI was 10:1,we added the bran first and stir-fried until smoking then added FAI over medium heat until the surface of FAI turns dark,so we obtain the fried FAI.The optimum chromatographic conditions were as follows:Alltima TMC₁₈ column（250 mm×4.6 mm,5μm）;mobile phase:acetonitrile-0.1%formic acid（20:80）;flow rate:1.0 m L/min;detection wavelength:284 nm;injection volume:10μL;column temperature:30℃,the content of naringin,hesperidin and neohesperidin in FAI decreased after stir frying with bran.2.After 21 days of continuous stimulation,compared with BG,the mental state of the rats was poor,the activity was low,the hair color was withered and yellow,and the body weight,gastric emptying rate and intestinal propulsion rate were significantly decreased（P<0.01）.After administration for 14 days,compared with MG,the weight of rats in PG and FFAIWB increased significantly（P<0.01）,the contents of GAS and MTL increased significantly（P<0.01）,and the contents of SS decreased significantly（P<0.01）.compared with MG,the body weight increased（P<0.05）,the secretion of GAS and MTL increased（P<0.05）,and the secretion of SS decreased（P<0.05）in RFAI.The results indicated that compared with raw FAI,fried FAI with bran could improve gastrointestinal motility,increase gastrointestinal hormone secretion and improve the general state of FD rats.3.Compared with BG,the m RNA and protein expressions of endoplasmic reticulum stress related factors such as IRE1,GRP78 were significantly increased in MG（P<0.05）,and the m RNA expression of JNK was significantly increased（P<0.05）.Compared with MG,the m RNA and protein expressions of IRE1 and GRP78 were significantly decreased,and the m RNA expression of JNK was decreased in PG and FFAIWB（P<0.05）.Compared with BG,the expression of Bcl-2 protein in the gastric tissue of rats in MG decreased while the expression of Bax protein increased（P<0.05）.Compared with MG,the expression of Bcl-2protein in the gastric tissue of rats in the PG and the FFAIWB increased and the expression of Bax protein decreased（P<0.05）.4.16S r DNA sequencing technology was used to analyze the intestinal flora of rats.The results showed that the diversity and abundance of intestinal flora decreased in FD rats.At the level of phylum,compared with BG,the relative abundance of Firmicutes reduced while the abundance of Bacteroidetes,Spirochaetes increased.According to the LEfse analysis and related literature we speculated that Prevotellacea,Lachnospiracea and Ruminococcaceae might be closely related to the occurrence of functional dyspepsia and the the structure of intestinal flora in FD rats can be regulated by stir fried with wheat bran FAI.Conclusion:After stir fried with bran,the contents of total flavonoids such as naringin,hesperidin and neohesperidin decreased significantly in FAI.Through pharmacodynamics investigation we found that RFAI and FFAIWB decoction could increase gastric emptying rate and intestinal propulsion rate,regulate gastrointestinal hormone secretion then improve the general state of FD rats.The mechanism may be related to inhibiting the expression of ER stress related factors such as IRE1,GRP78 and JNK in gastric tissue,relieving ERS then reducing the expression of apoptotic protein so as to enhance gastric motility.The changes of relative abundance of Prevotellacea,Lachnospiracea and Ruminococcaceae in intestinal microflora may related to FD,and the regulation effect of FFAIWB on intestinal microflora structure of FD rats is better than that of raw products.In conclusion,although the content of flavonoids in FAI decreased after stir frying with bran,it could alleviate FD related symptoms by regulating gastrointestinal hormone secretion,inhibiting endoplasmic reticulum stress and regulating intestinal flora structure and abundance.更多还原