Study On The Effect Of Modified Ginseng Wumei Decoction And Its Flavor Decomposition Formulas On Colon Differential Gene Expression In Diarrhea Model Rats Based On The Theory Of " Five Flavors Combination And Transformation "

OBJECTIVE: Based on the “Five flavors combination and transformation” theory,the whole gene chip technology was used to explore the molecular regulation mechanism of modified Ginseng-Mume Decoction(Sour-sweet herbs nourishing yin)on the colon differential genes in diarrhea model rats.METHODS: Eighty-four young age SD rats were fed ad libitum for 2 days of acclimation.A total of 12 rats were randomly selected as the normal group(group A)and given by intragastric administration with normal saline(30 ml/kg,1 time/d).The remaining 72 rats were for the model group,and by reference to the literature,to make the diarrhea model by the combination of bitter-cold laxatives and swimming exhaustion.25% senna leaf decoction was intragastrically administered(20ml/kg,1 time/d);after 30 minutes of daily gavage,the rats were swam in water(water depth 40 cm,water temperature 30°C,tail load with about 5% of self weight lead),until exhausted(rats nose tip under water for 5 seconds),then remove and dry.Continuous molding 21 days.After the successful evaluation of the model,the model group was randomly divided into the model group(group B),the western medicine group(group D),the whole prescription group(the modified Ginseng-Mume Decoction,group E),the de-sweet herbs group(the de-sweet herbs modified Ginseng-Mume Decoction,group F),the de-sour herbs group(the de-sour modified Ginseng-Mume Decoction,G group),the de-piquant herbs group(the de-piquant modified flavored Ginseng-Mume Decoction,group H),12 in each group.They were respectively given by intragastric administration with normal saline(30 ml / kg,once a day),5% Medilac-Vita(0.7 g / kg,once a day),the whole prescription modified Ginseng-Mume Decoction,the de-sweet modified Ginseng-Mume Decoction,the de-sour modified Ginseng-Mume Decoction,the de-piquant modified Ginseng-Mume Decoction(The dosage of each Chinese medicine group was 35 g / kg,once a day),and continuous intervention was made for 7 days.To observe the general situation of rats in the process of modeling and intervention.The rats were killed 7 days after intervention,and the samples were taken for examination.(1)Colon tissue was fixed with 10% formaldehyde fixative,routine HE staining,and histopathological morphology was observed under light microscope.(2)The colon tissue was fixed by RNA preservation solution,and after RNA extraction,quality testing,purification and amplification,the Agilent rat whole gene 4*44K chip was used for hybridization scanning,and the original data of the chip was normalized and statistically screened by the differentially expressed genes between groups(FC?2,p<0.05);Bioinformatics analysis of differential genes was carried out by GO function and KEGG pathway enrichment analysis method,and the differential gene function was preliminarily explained.(3)Real-time quantitative PCR was used to verify the results of the chip,and the amplification results were statistically analyzed.RESULTS:(1)Pathological observation of colon: The western medicine group and the whole prescription group can effectively alleviate the general manifestations and diarrhea of diarrhea rats,and can significantly improve colonic mucosal ulcer,congestion,edema,inflammatory infiltration and other reactions in diarrhea rats,having a certain damage repair ability.For the colonic mucosal damage in the rats,the de-sweet medicine group,the de-sour medicine group and the de-piquant medicine group were with weaker damage repair ability but the degree of intestinal mucosal damage was lighter than that in the model group.(2)Whole-genome microarray experiment and bioinformatics analysis:(1)Compared with the normal group,the model group obtained 34 differential genes,including 10 up-regulated genes and 24 down-regulated genes;GO enrichment obtained 123 items,including 92 biological process items(significant enrichment(p <0.05)44 items),mainly involved in ion transport,ion homeostasis,developmental regulation,cell differentiation,signal transduction,cell proliferation,etc;KEGG pathway enrichment to obtain 21 pathways,mainly involving hypertrophy Cardiomyopathy(HCM),myocardial contraction,MAPK signaling pathway,neuroactive ligand-receptor interaction,calcium signaling pathway,etc.(2)Compared with the model group,the western medicine group obtained 16 differential genes,including 6 up-regulated genes and 10 down-regulated genes;GO enrichment obtained 5 items,including 2 biological process items(no significant enrichment),involving cell process regulation;KEGG pathway enrichment acquiresd 2 pathways involving olfactory transduction and RNA degradation.(3)Compared with the model group,the whole prescription group obtained 51 differential genes,including 17 up-regulated genes and 34 down-regulated genes;GO enrichment obtained 125 items,including 80 biological process items(significant enrichment 12 items),mainly involving damage response,signal transduction,and transcriptional regulation;KEGG pathway enrichment obtained 21 pathways,mainly involved in neuroactive ligand-receptor interaction,olfactory transduction,lysine degradation,protein digestion and absorption,taste transduction,calcium signaling pathways,etc.(4)Compared with the model group,the de-sweet medicine group get 45 differential genes,including 19 up-regulated genes and 26 down-regulated genes;GO enrichment obtained 72 items,including 35 biological process items,mainly involving reactions to other organisms,response to external biostimulation;KEGG pathway enrichment obtained 37 pathways,mainly involved in phagocytosis,olfactory transduction and so on.(5)Compared with the model group,the de-sour medicine group obtained 145 differential genes,including 71 up-regulated genes and 74 down-regulated genes;GO enrichment obtained 411 items,including 306 biological process items,mainly involved in Negative regulation of lipid metabolism,response to interferon-?,etc;KEGG pathway enrichment obtained 53 pathways,mainly involving primary immunodeficiency,cytoplasmic DNA sensing pathway,etc.(6)Compared with the model group,the de-piquant medicine group obtained 126 differential genes,including 52 up-regulated genes and 74 down-regulated genes;574 items were enriched by GO,including 439 biological process items,mainly involving androgen biosynthesis process,C21 steroid hormone biosynthesis process,etc;KEGG pathway enrichment obtained 73 pathways,mainly related to steroid hormone biosynthesis,tryptophan metabolism,etc.Compared with the model group,the biological functions involved in the different genes of each disassembled prescription group,the most is the de-piquant medicine group,following is the de-sour medicine group,the least is the de-sweet medicine group,and all the three groups are different from the whole prescription group.Compared with whole prescription group,the difference genes was the least in the depiquant medicine group.(3)RT-PCR validation experiments: Nlrp6,Tmem66,Gng10,Shc1,Ctnnb1,Trpm7,Vamp7,and Gnas were screened as validation genes.The overall expression trend of the validation genes after amplification was in good agreement with the results of the gene chip experiment.After amplification of the validation genes,compared with the normal group,the difference of Gng10 and Trpm7 in the model group were statistically significant(p<0.05),and the expression was up-regulated;compared with the model group,the difference of Nlrp6 and Tmem66 in the whole prescription group was statistically significant(p<0.05),and the expression was down-regulated;all of them are consistent with the results of the chip experiment.CONCLUSIONS:(1)The modified Ginseng-Mume Decoction has a repairing effect on colonic mucosa damage of diarrhea model rats.(2)Reflecting diarrhea has the characteristics of multiple genes and multiple pathways in pathogenesis.Down-regulation of CHRNA7 affects calcium ion regulation,downregulation of CACNA2D3 and up-regulation of IL1R2 affect MAPK signaling pathway,which may be involved in the development of diarrhea.(3)Preliminarily proving the modified Ginseng-Mume Decoction(Sour-sweet herbs nourishing yin)can enhance qi while nourishing fluid and anti-diarrhea by regulating the expression of colon differential genes in diarrhea model rats.The modified GinsengMume Decoction may play a anti-diarrhea effect by down-regulating F2RL3 and GHR affecting the neuroactive ligand-receptor interaction pathway,up-regulating SUV39H2 affecting lysine degradation pathway,and down-regulating XPNPEP2 affecting protein digestion and absorption pathways.(4)Combined with the previous research of the fingerprint of modified Ginseng-Mume Decoction,The effect of which enhancing qi while nourishing fluid and anti-diarrhea mainly through the combination of sour medicine & sweet medicine to realize,and emboding the complex action characteristics of traditional Chinese medicine compound with multi-component,multi-gene,multiple pathways etc.(5)RT-PCR verification experiments confirmed that the results of gene chip experiments are reliable.Gng10,Trpm7,Nlrp6 and Tmem66 have certain potential value as molecular targets for the pathogenesis and treatment of diarrhea.