Study On The Mechanism Of Fried Fructus Aurantii With Bran Based On Endoplasmic Reticulum Stress And Intestinal Flora In Rats With Functional Dyspepsia

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.