Experimental Study On The Treatment Of Reflux Esophagitis With Morinda Citrifolia And Stem Cell Transplantation

IntroductionGastroesophageal reflux disease (GERD) is a kind of disease in which the contents of stomach and duodenum flow back to th e esophagus,causing heartburn,acid regurgitation,and thermalgia behind the sternum.Reflux esophagitis (RE) is a type ofGERD.At present,the treatment options include medication and surgery.The disadvantages of medical therapy are easy to relapse,diffcult to determine the course of treatment,and many side effects.The surgical methods are limited because of their complications and sequelae.Therefore,it is of great significance to find a new alternative therapy.Bone marrow derived mesenchymal stem cells (BMMSC) transplantation,as a new biological therapy in recent years,has been widely valued.It has the advantages of small immunogenicity,no side effects of drugs,easy access and amplification.Studies have shown that injecting stem cells into the myometrium of the rat esophagus is helpful for the treatment of GERD.However,it is still unknown whether BMMSC can naturally circulate to the injured region of the esophagus and reconstruct the injured esophageal mucosa in RE model.Some studies have confirmed that stem cells can promote intestinal metaplasia and other pathological changes in the RE model.Can these metaplasia cells not only come from the progenitor cells in the base of esophagus,but also from the circulating BMMSC?If possible,stem cell therapy has potential risks due to the existence of such a pathophysiological process as inflammation-metaplasia-adenocarcinoma.It is necessary for us to find a way to inhibit the development of columnar metaplasia during stem cell transplantation and improve the safety of stem cell transplantation.The purpose of this study is to reveal the repair effect of BMMSC,and to evaluate whether it can differentiate into squamous epithelium and metaplasia columnar epithelium to participate in the repair.At the same time,the effect and mechanism of Morinda citrifolia on BMMSC differentiation will be studied,and a new idea for the treatment of GERD may be provided.In traditional Chinese medicine,Morinda Officinalis is a dicotyledonous plant,Rubiaceae.It has another name,Rabbit-in testin es and Chicken-Intestines-Wind,which is produced in Fujian,Guangdong,Hainan,Guangxi and other provinces of tropical and subtropical areas.The plant grows in sparse,dense forests and thickets in the mountains,often climbing on shrubs or tree trunks.It is also distributed in Indochina Peninsula.Morinda Oficinalis How belongs to the traditional Chinese medicine of invigorating Yang.Morinda Officinalis How is the dry root of Morinda officinalis,a perennial Liana of Rubiaceae.The natural flavor of Morinda Officinalis How is spicy,sweet and mild.It bellongs to the kidney meridian.Efficacy of Morinda Officinalis How include:Invigorating Kidney and helping Yang,dispelling wind and dehumidification.The functions of Mornda Officinalis How include:1.Enhancing immune function;2.Anti inflammation;3.Improving reproductive system function.Morinda citrifolia L.,a close relative of Morinda Officinalis How,is an evergreen shrub or small tree of Morinda in Rubiaceae.It is mainly distributed in Taiwan,Hainan and Xisha Islands.It is also called Noni in the above areas.It has strong viability,edible and medicinal value,and is rich in a variety of nutrients.It is also distributed in Hawaii and Tahiti islands abroad.Morinda citrifolia L.,Noni,is a tropical plant,which grows in Hawaii and Tahiti islands and be rich in various nutrients.It has been proved that its effective components can inhibit colitis and maintai in the integrity of intestinal mucosa.Scopoletin,which is contained in the extract of Morinda citrifolia,has also been proved to have the effect of inhibiting esophageal inflammation.All these studies indicate that Morinda officinalis has similar anti-inflammatory effeect with Morinda Officinalis How.Other studies have confirmed that rutin and other components in the extract of Morinda citrifolia have the effect of promoting cell differentiation,but there is no report that Morinda Officinalis How has this effect.It has been confirmed that the differentiation of stem cells depends on the microenvironment which contains them.So we suppose that Morinda citrifolia Extract(MCE) may be able to improve the esophageal microenvironment of GERD patients through its own anti-inflammatory and antioxidant effects,create a more favorable microenvironment for stem cells to differentiate in the right direction,and promote them to differentiate more to normal esophageal epithelial cells,rather than to columnar epithelial cells,thus delaying Barrett esophagus (be) and even esophageal adenocarcinoma Adenocarcinoma,EAC).Therefore,MCE combined with BMMSC transplantation was selected to treat GERD,and the synerrgistic effect of MCE and BMMSC on GERD was evaluated.If it can reduce the differentiation of columnar epithelium,it will help to improve the safety of stem cell transplantation,provide the theoretical basis for MCE combined with stem cell transplantation in the treatment of GERD,and finally contribute to its clinical application.This topic is divided into three parts:Part ?:Comparison of rat model of gastroesophageal reflux disease Objective:To evaluate the sensitivity of various GERD models to drug intervention and the incidence of intestinal epithelial differentiation.Methods:To compare the perfusion model of exogenous acidified pepsin (AP) at different time interrvals,Esophagogastric Jejunostomy (EGJ),Esophageal Jejunostomy(EJ),Esophageal Jejunostomy with Gastrectomy (EJ/TG).The macroscopic score of the model was evaluated,including hyperemia,edema,erosion,ulcer,intramural or intracavitary hemorrhag;the microscopic score was also be evaluated,including epithelial loss (separation,erosion,ulcer),reactive epithelial change (basal hyperplasia,papilloma,mitosis,in situ keratosis,incomplete keratosis),vascular change (hyperemia,edema,hemorrhage,angiopathy),inflammation (inflammatory cell aggregation and diffusion);pH value of lower esophageal segment was measured by pH electrode;submucosal blood flow was measured by ultrasound;proliferation of mucosal basal cells was studied by anti-Ki-67 immunohistochemistry;expression of Cdx-2,TFF1 and TFF2 was studied by immunohistochemistry.The improvement value of macro and micro scores after drug treatment was evaluated in various models,and the sensitivity of varous models to drug intervention was evaluated.The effects of various methods on the metaplasia and dysplasia of columnar epithelium was evaluated.Results:In the AP perfusion model,the scheme of 45 min/bid.was used to construct the model,which lasted for 5 days.HE staining indicated that the model caused inflammation,erosiion and ulcer,but maintained the epithelial and contributed to different degree of epithelial reactivity changes.No columnar metaplasia was observed in all AP perfusion models.In the AP perfusion model,60 min/bid.for 3 days and 45min/bid.for 5 days were more sensitive to drug intervention than all the surgical GERD model.Compared with other methods,EGJ could cause the highest incidence of columnar metaplasia,which was 39.4%.Conclusion:Both the external AP perfusion scheme and the surgical modeling scheme can be used to evaluate the effectiveness of the drugs.The model constructed by the scheme of exogenous AP perfusion for 45 min/bid and lasting for 5 days has the highest sensitivity to drug intervention,which is suitable for evaluating the therapeutic effect of MCE combined with BMMSC.The proportion of the metaplasia of columnar epithelium is the highest in the endogenous EGJ model.It is suitable to study the effect of MCE on the differentiation of BMMSC in the EGJ model.Part ?:The role and mechanism of MCE in promoting the differentiation of BMMSC into esophageal epitheliumObjective:To study whether BMMSC can differentiate into esophageal epithelial cells(EEC) under the condition of co-culture with EEC,and whether this differentiation is related to Wnt/?-catenin signaling pathway,and to study the effect of MCE on the differentiation of BMMSC and its mechanism.Methods:BMMSC and EEC of rats were co-cultured,and the expression of Pan-CK,a surface marker of EEC,in BMMSC was detected.After co-culture with EEC,Western blot was used to detect) GSK-3? and ?-catenin in BMMSC.The effect of Wnt/?-catenin signal activatorr LiCL on phosphorylated GSK-3? and P-catenin was detected by Western blot and Pan-CK expression was detected by immunocytochemistry.MCE was prepared and identified by HPLC.MCE was used in three cases:BMMSC co-cultured with EEC,BMMSC cultured in keratinized medium and BMMSC cultured in common medium independently.The effect and mechanism of MCE on the epithelial differentiation of BMMSC were studied.The cytotoxicity of MCE was detected by commercial kit,EGF was detected by ELISA,Pan-CK was detected by cytochemical staining,and the effect of MCE on the exprelssion of CK18,CK19,EMA,Wnt3a,?-catenin,EGF,CDX2 and other genes was detected by qRT-PCR.Western blot was used to detect the phosphorylation of Akt and GSK-3? related to Wnt/?-catenin signaling pathway.Immunofluorescence combined with laser confocal microscopy was used to detect the effect of MCE on the?-catenin by nuclear migration and transcription activity.In addition,DKK-1 (inhibitor of Wnt) and LY294002 (inhibitor of PI3K) were added respectively to compare the above detection targets.Results:BMMSC co-cultured with EEC could obtain EEC phenotype.During this process the main members of Wnt/p-catenin signaling pathway named phosphorylated GSK-3? and p-catenin increased,and the activator LiCL increased the expression of phosphorylated GSK-3? and ?-catenin,and enhanced the expression of Pan-CK.It is suggested that the activation of Wnt/p-catenin signaling pathway promotes the epithelial differentiation of BMMSC.The active components of MCE were identified as kaempferol-3-o-rutin and rutin.MCE did not show cytotoxicity,0.0038% (w/v%)concentration could increase EGF content in the co-culture system,enhance the expression of Pan-CK,upregulate the expression level of CK18,CK19,EMA,Wnt3a,?-catenin,EGF genes,but had no effect on CDX2 gene.In Wnt/p-catenin signaling pathway,MCE can increase the phosphorylation level of Akt and GSK3?,as well as the nuclear transport and transcription activity of ?-catenin,which will be weakened by the addition of Wnt inhibitor DKK-1 or PI3K inhibitor LY294002.Conclusion:Co-culture with EEC can promote the differentiation of BMMSC by activating Wnt/?-catenin pathway.MCE with a minimum effective dose of 0.0038%(w/v%) can enhancei the epithelial differentiation of BMMSC by promoting EGF secretion and PI3K/Akt-dependent activation of Wnt/?-catenin signaling pathway.MCE combined with BMMSC transplantation may be a new treatment for GERD esophageal injury.Part ?:Study on the anti-inflammatory and BMMSC differentiation promoting effects of MCE in rratsObjective:To study the anti-inflammatory effect of MCE in GERD rat model and the effect of MCE on the differentiation of BMMSC in esophageal microenvironment.Methods:Raw 264.7 cells were used to evaluate the cytotoxicity of MCE and the MCE effeect on inflammatory markers (NO,PGE2,IL-1(3).In the first part,the AP perfusion model with the best sensitivity to drug intervention was selected to evaluate the anti-inflammatory effect of MCE in rat s and the repair effect of MCE combined with BMMSC on GERD.SD rats were divided into 8 groups:normal control group,RE control group,rabeprazole group,single MCE group (three dose subgroups),siingle BMMSC group,MCE combined BMMSC group.To evaluate gastric volume and pH value of gastric juice,analyze mucosal injury index,detect TNF-?,IL-1,?IL-6 and MCP-1 in serrum by ELISA,and detect mRNA expression of TNF-?,IL-1?,IL-6 and PAI-1 by qRT-PCR.EGJ model was selected to evaluate the effect of MCE on the differentiation of BMMSC in esophageal environment.BMMSC labeled with GFP were transplanted after operation,and the differentiation of BMMSC was detected by immunohistochemistry.Pan-CK and CDX2 were detected by confocal laser microscopy to evaluate the effect of MCE on the differentiation of BMMSC.Results:With the increase of MCE concentration,esophageal inflammation decreased in a dose-dependent manner.In MCE group,the decrease of serum inflammatory markers was detected by ELISA,and the decrease of inflammatory marker mRNA was detected by qRT-PCR.The tracing results of GFP in the BMMSC showed that BMMSC could repair the damaged esophageal epithelium in GERD.Under the intervention of MCE,more BMMSC differentiated into squamous cells rather than columnar cells.Conclusion:The anti-inflammatory effect of MCE can be used to treat RE in rats,and the combination of MCE and BMMSC transplantation can produce synergistic effect.MCE can promote the differentiation of BMMSC into normal squamous epithelium in esophageal microenvironment,reduce the differentiation of columnar epithelium,improve the safety of BMMSC transplantation.The combination of MCE and BMMSC become a treatment that may delay or prevent the progression of BE in rats.