Mechanism Of Jianpi Huayu Jiedu Therapeutic Method In Regulating The NF-κB Signaling Pathway In The Migration Of "Inflammatory-cancer" Transition Cells

BackgroundAccording to Correa hypothesis,the pathological evolution of gastric cancer is "normal gastric mucosa→ chronic non-atrophic gastritis→ chronic atrophic gastritis→intestinal epithelial dysplasia",and it is finally come up into gastric adenocarcinoma,where in the higher risk of cancer,including intestinal metaplasia,dysplasia,thus being collectively referred to gastric precancerous lesions(GPL).Based on this,Blocking or delaying GPL progression to gastric cancer,becomes measures to prevent gastric cancer."Chronic gastritis-atrophic gastritis-dysplasia-gastric cancer" is called "inflammatory-cancer" chain,wherein non-controllable inflammation plays an important role in GPL "inflammatory-cancer"transition,so anti-inflammatory is important in GPL treatment particularly.At present,modern medicine drugs targeting GPL is still blank.Although clinically used drug protein,Helicobacter bacteria,COX inhibitors,etc.,can delay chronic atrophic gastritis to a certain extent,it also exists long time to take medicine and unstable efficacy.However,traditional Chinese medicine exists unique advantages for the treatment of GPL while its experiencetar geting GPL is not summarized.Therefore,this paper regarded stomach "inflammatory-cancer" into an entry point.By exploring the mechanism of Jianpi Huayu Jiedu therapeutic method in regulating the NF-κB signaling pathway in the migration of cells,we discussed the molecular mechanisms of GPL pathogenesis and the efficacy of JPHYJD recipe in intervening and even reversing the"inflammatory-cancer" transition.1.The C57 mouse Balb/c which is the most sensitive to carcinogenic factor are used,and the N-methyl-N’-Nitro-N-Nitroguanidine(MNNG)solution is free to drinkto establish the GPL model.By observing the pathological changes of gastric mucosal tissues,the expression of GPL molecular markers(Ki67,P53),body weight and feeding of the mice,screened and identified the model of "inflammatory-cancer" transition;2.The improving role of the traditional Chinese decoction JPHYJD in attenuating the inflammation and the molecular mechanism by inhibiting NF-κB signal pathway on the"inflammatory-cancer"transition are discussed;3.Based on the previous studies that JPHYJD recipe blocked the gastric "inflammatory-cancer" transition in mice though inhibited NF-κB activity,the early migration of the gastric mucosa cells were observed in vivo and in vitro,which aimed at exploring the role of JPH YJD recipe in the intervention and even reverse of the stomach "inflammatory-cancer" transition from the perspective of the downsteam of NF-κB signal pathway.MethodsFirstly,exploring the modeling conditions of GPLCs cell model from the perspective of different model time,concentration,et al,thus innovate the modeling method of GPLCs cell model.At the same time,gastric cancer cell EMT model is constructed to detect the effects of JPH YJD decoction at the stage of gastric cancer EMT.The cell survival rate,EMT-related gene expression and scratch healing experiments are detected to observe cell migration,thus clear the intervention effect of Jianpi Huayu Jiedu therapeutic method representative decoction Weipixiao(WPX)on cell migration;Based on theJianpi Huayu Jiedu therapeutic method representative decoction WPX combined with GPL "Deficiency toxicity and blood stasis" pathogenesis,we come up with the JPH YJD recipe and analyse the main active ingredients of JPH YJD recipe through high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)analysis.The retention duration and ion chromatogram of the reference compound are compared to JPHYJD recipe,therefore identify the main components of the JPHYJD decoction and establish quality control standards of the JPHYJD recipe;The GPL "inflammatory-cancer" transition mouse model are constructed though chemical mutagenic,after intervention of JPHYJD recipe,observe changes in stomach mucosa structure,detect cancer markers,inflammatory factors,inflammatory cell infiltration and the activation of NF-κB signal pathway;The chemical mutagen MNNG is constructed to induce GPL "inflammatory-cancer"transition in vivo and in vitro,then observe changes in gastric mucosal structure,detect the cell survival,cell migration and related protein expression after intervention of JPHYJD decoction.ResultsThe morphological changes exists in the MNNG induced gastric"inflammatory-cancer" transition cell model,such as cellular polarity lost,arrangement disorder,and company with pseudo-foot formation.In molecular mechanism,expression of EMT related molecular markers changed in model group cells,such as Vimentin(epithelial,interstitial phenotype marker),and MMP2.Jianpi Huayu Jiedu therapeutic method representative decoction WPX can decrease in EMT-related gene expression to a certain extent,and inhibit cell migration,but the efficacy is not significant;normal gastric mucosa epithelial cells and gastric cancer did not have significant invasive migration.2.HPLC-MS analysis was firstly performed to determine the main components in JPHYJD by matching retention times and mass spectra of the standard compounds with those of JPHYJD.The main components in JPHYJD,including notoginsenoside R1,calycosin-7-glucoside,astragaloside A,andadenine-9-β-D-ribofuranoside,were identified by comparing retention times and ion chromatograms with those of the reference compounds.Moreover,the fragment ion that obtains the highest intensity in the full MS spectra of the precursor ion was extracted for quantification,the amount of each compound in JPHYJD recipe was calculated after correlating the peak area of the analytes with the calibration curves of the standards,all calibration curves of reference compounds showed good linearity.Both notoginsenoside R1 and calycosin-7-glucoside were found to be the most abundant compounds,further suggesting that the compounds should be considered as chemical markers of JPHYJD.3.H&E staining and immunohistochemical staining(IHC)results showed that MNNG can induce key pathological changes of gastric"inflammatory-cancer" transition in stomach mucosa,such as atrophy of gastric mucosa,intestinal metaplasia,inflammatory cell infiltration and dysplasia,suggesting gastric "inflammatory-cancer" transition mouse model is successfully constructed.However,JPHYJD recipe can alleviate MNNG-induced gastric "inflammatory-cancer" transition,including gastric mucosal pathological tissue structural changes,pro-inflammatory Th1 cells infiltration and gastric mucosal epithelial tissue cancer markers expression(Ki67,p53),suggesting that JPHYJD recipe can protect the gastric mucosa from inflammation-mediated damage.4.Western-blot results show that JPHYJD recipe can reverse MNNG induced NF-κB/IκB-α activation,COX-2,NADPH oxidase family NOX2 and NOX4 protein expression,qPCR test results showed that JPHYJD recipe can significantly alleviated MNNG-induced inflammatory factors(IL-6,TNF-α).Compared with vitamin B12(VitB12)group,high-dose of JPHYJD recipeexist stronger anti-inflammatory activity.5.In addition,NF-κB activation can induces "inflammatory factor storm",which improved protein activation of early cell migration during the gastric"inflammatory-cancer"transition process.Western-blot results show that MNNG can induce activation of early cell migration related protein expression in vivo and in vitro,such as E-cad,N-cad,etc.,and JPHYJD recipe pretreatment can significantly inhibit the activation of these proteins,thereby interfere with the progress of the gastric"inflammatory-cancer" transition.ConclusionGPLCscellexisted EMT,suggesting GPLCs cell may participate in early metastasis of gastric cancer.Jianpi Huayu Jiedu Chinese medicine WPX can control the expression of EMT related genes to a certain extent,therefore inhibit cell migration,but the efficacy is not significant.Based on the phenomenon exist aboved,Chinese medicine decoction in Jianpi Huayu Jiedu needs to be further optimized.2.Both notoginsenoside R1 and calycosin-7-glucoside were found to be the most abundant compounds,further suggesting that this two compounds can be considered as chemical markers of JPHYJD recipe.3.MNNG can induce gastric"inflammatory-cancer" transition in vivo and in vitro.This animal model was successfully established attributed to selection of Balb/c mice which sensitive to carcinogens,and it can stably simulate the pathological changes of gastric"inflammatory-cancer" transition.4.JPHYJD recipe can intervene the process of gastric "inflammatory-cancer" transition by mediating the "inflammatory factor storm" caused by NF-κB in gastric mucosa.At the same time,it may inhibit cell migration by inhibiting its cascade reaction,so as to exert the potential of controlling and even reversing the effect of gastric "inflammatory-cancer"transition,and then prevent the occurrence of gastric cancer.In conclusion,we successfully constructed the gastric "inflammatory-cancer" transition model mice.JPHYJD recipe may regulate NF-κB signaling pathway,thus inhibit the cascade reaction of "inflammatory factor storm" caused by its activation.Therefore,based on the intervening effect of pathological changes such as gastric mucosal atrophy,intestinal metaplasia and dysplasia in mice,JPHYJD recipe can alleviate the early migration of gastric mucosal epithelial cells induced by MNNG thus exert the potential of treating GPL and preventing gastric cancer.