The Action Mechanism Of Patchouli Alcohol In Treating Intestinal Mucositis

Research backgroundPogostemon cablin(Blanco)Benth,a traditional plant in China used for treating gastrointestinal disorders by eliminating dampness,preventing vomiting and relieving summer-heat.Patchouli alcohol(PA),is the most abundant and popular tricyclic sesquiterpenes component of Patchouli oil.Reseachs have found PA processes multiple bioactivities,including gastrointestinal protection,anti-inflammation and maintenance of gut microbiota.The study of gastrointestinal protection is the most important focus among these bioactive effects.Intestinal mucositis(IM).also known as intestinal mucositis loss,is the main dose-limiting adverse reaction in tumor patients during chemotherapy.The main clinical manifestations are a series of gastrointestinal reactions,such as nausea,vomit,diarrhea,loss of appetite and indigestion.IM,characterized by high incidence and great harm,is often leading to ulcer formation.bleeding,diarrhea,pain and other symptoms,which seriously affects the treatment and life quality of tumor patients and increases the probability of infection and mortality,as well as affecting the course of chemotherapy.Therefore,to improve IM and related gastrointestinal disorders caused by chemotherapy has a high clinical application value and social value for improving the life quality and prognosis of tumor patients.Pogostemon cablin is a traditional medicine for gastrointestinal diseases in China.In the previous study on the anti-ulcerative colitis of patchouli alcohol,patchouli alcohol could alleviate symptoms of intestinal inflammation,such as diarrhea,weight loss,etc.Further studies have found that patchouli alcohol promotes the recovery of intestinal barrier function.The clinical symptoms of intestinal mucositis are similar to ulcerative colitis,and impaired intestinal barrier function is an important cause of intestinal mucositis.Therefore,we speculate that patchouli may have active effect on the treatment of intestinal mucositis.This experiment would explore the mechanism of action of patchouli alcohol against intestinal mucositis from the perspective of intestinal barrier function,and provide a scientific basis for the treatment of intestinal mucositis with patchouli alcohol.MethodsExperimental animals were randomly divided into normal group,model group,positive group and administration group.From first to fourth day in the period of experiment,rats in the normal group were administrated normal saline by intraperitoneal(i.p.)injection while other rats were injected(i.p.)5-fluorouracil(5-FU,30 mg/kg)in each day to establish the intestinal mucositis(IM)model.During the experiment,the rats in normal and model groups were both received normal saline,while rats in positive group and PA groups were orally administrated CBL(400 mg/kg)and PA(10,20 and 40 mg/kg,respectively)for 7 days.The defecation status,weight and food intake were observed and recorded every day.At the end of experiment,the efficacy of patchouli alcohol in the treatment of intestinal mucositis was initially evaluated by considering the changes of weight,food intake,diarrhea scores and histopathology.The small intestinal contents of every experimental sample were collected for extracting total DNA,and using 16S rDNA V4 sequencing analysis to detect PCR produces sequences and then splice into the Tags.Species classification?abundance analysis?OTU analysis?species evolution and cluster analysis were used to observe the difference among all the groups according to Tags.All results were help to investigate the activity of PA on gut microbial diversity and flora distribution.The small intestinal tissues were used to detect following indicators.The expression of goblet cells and mucin-2 were detected by PAS and IHC,respectively;the mRNA levels of MLCK,ZO-1,Occludin and Claudin-1 in small intestinal tissues were detected by RT-PCR;the related proteins(NF-?B p65?p-p65?I?B??p-I?B? ZO-1?Occludin?Claudin-1)were evaluated by using Western blot;the relevant inflammatory and oxidative indicators,including TNF-??IL-1??IL-4?IL-6?IL-10?slgA and MPO,were detected via ELIS A.ResultsThe routine results showed that body weight and feed consumption of rats in model group decrease and their diarrhea became more and more serious.CBL administration could increase significantly the rats' body weight and feed intake,as well as improving diarrhea in the later stage of the experiment.PA has the similar effects which significantly relieved the weight loss and diarrhea of the experimental rats,recover drinking and feeding.Histopathology results showed 5-FU seriously damaged the small intestine,caused tissue necrosis,mucosal atrophy and incomplete glands.CBL administration could protect small intestine,which have hardly necrosis area.The protective effect of PA on rats' small intestine was dose-dependent.The rats in 40 mg/kg PA group had relatively complete tissues and well-ranged glands,while the small tissues were damaged and the glands were disordered in 10 mg/kg PA groups.Gut microbiota analysis results also verified that 5-FU could destroy the homeostasis and compositional diversity of intestinal flora in rats.CBL and PA could increase the number of OTU and restore the composition of flora in IM rats.What's more,PA could significantly inhibit the growth of pathogenic bacteria(e.g.bacteroides,helicobacter,Parabacteroides)and promote the growth of dominant bacteria(e.g.bifidobacterium,lactobacillus).PAS and IHC staining results showed 5-FU could significantly decrease IOD of goblet cells and mucin-2 in rats,which could be increased effectively in positive group.The IOD enhancement of goblin cells and mucin-2 in PA group was dose-dependent.40 mg/kg PA group had higher IOD than positive group,which means 40 mg/kg PA had best effect in increasing expression of goblet cells and mucin-2.RT-PCR showed 5-FU could inhibit mRNA expression of MLCK?ZO-1?Occludin and Claudin-1,while CBL could reverse the trend to restore above mRNA expression.40 mg/kg PA had the best recovery effect and significantly increase the mRNA expression of MLCK?ZO-1?Occludin and Claudin-1.Western blot suggested 5-FU could activate TLR2/MyD88/NF-?B pathway and destroy intestinal epithelial cells tight junction proteins,including ZO-1?Occludin?Claudin-1.CBL and PA could effectively inhibit TLR2/MyD88/NF-?B related proteins expression and restore expression of ZO-1?Occludin?Claudin-1 in intestinal epithelial cells in IM rats.The effect of different PA groups was in a dose-dependent manner.Elisa results showed TNF-??IL-1??IL-6?slgA and MPO were overexpression and IL-4?IL-10 were low expression in model group.CBL and PA could reduce the expression of TNF-??IL-1??IL-6?sIgA and MPO,and increase the expression levels of IL-4?IL-10.In three dosage of PA groups,40 mg/kg PA had the most significant effect.ConclusionsIn summary,patchouli alcohol can effectively improve the symptoms of chemotherapy-induced intestinal mucositis rats,relieve intestinal inflammatory reaction and diarrhea,as well as recovering drinking and diet.Inner treatment mechanism was related with protecting intestinal barrier function,maintaining intestinal microflora Ecological stability,improving intestinal epithelial permeability and inhibiting TLR2/MyD88/NF-?B pathway.Above results suggested that patchouli alcohol has potential applications in intestinal mucositis caused by tumor chemotherapy.