Effect Of The Jianpi Huoxue Decoction Therapy On CAG Precancerous Lesions And Mechanism Study Based On Transcriptome Sequencing

Objective:Clinical symptoms,gastroscopy findings,and pathological efficacy of the observation of the Jianpi Huoxue Decoction in the treatment of precancerous lesions of chronic atrophic gastritis(CAG)of the spleen deficiency and blood stasis type were investigated.A cell model of precancerous lesions of gastric cancer(PLGC)was established by using Methylnitronitrosoguanidine(MNNG)to induce malignant transformation of human gastric mucosal epithelial cells(GES-1).The differentially expressed mRNAs and lncRNAs of MC cells before and after intervention with the Jianpi Huoxue Decoction were detected using whole transcriptome sequencing technology to explore the possible mechanism of action of the Decoction in treating precancerous lesions of CAG.Methods:1.Clinical StudyA total of 92 patients diagnosed with spleen deficiency and blood stasis type precancerous lesions of CAG,treated at Outpatient and Ward of General Internal Medicine Department of Jiangsu Provincial Traditional Chinese Medicine Hospital from April 2019 to June 2022,were collected.The patients were randomly divided into an experimental group and a control group using a digital randomization method,the experimental group had 3 cases of dropout and the final number of cases was 43,while the control group had 5 cases of dropout and the final number of cases was 41.The experimental group received the Jianpi Huoxue Decoction(Astragalus membranaceus 15g,Atractylodes macrocephala 10g,Coix seed 15g,Curcuma zedoaria 10g,Stir-fried fructus aurantii 10g,Fructus fritillaria 10g,Endothelium corneum gigeriae galli 10g,Chinese Sage Herb 15g,etc.,provided by Traditional Chinese Medicine Pharmacy of Jiangsu Provincial Hospital of Traditional Chinese Medicine),administered once daily,decoction in water 2 times,200 ml per dose,by decoction after breakfast and dinner.The control group received oral folic acid tablets(5 mg,Batch No.:H32023302,produced by Changzhou Pharmaceutical Factory Co.,Ltd.),three times daily,1 tablets per dose,for a duration of 12 weeks.The clinical symptoms,gastroscopy findings,and pathological scores of the two groups before and after treatment were observed and statistically analyzed.2.Experimental Study2.1 Inhibition effect of Jianpi Huoxue Decoction on the growth,migration and invasion of MNNG-causing GES-1 malignant transformed cells(MC cells)in vitroFirst,GES-1 cells,human gastric mucosal epithelial cells,were established as a blank control.The effects of MNNG,Jianpi Huoxue Decoction and folic acid on the viability of GES-1 cells were evaluated using the MTT assay to determine suitable intervention concentrations and durations.MNNG was used to induce malignant transformation of GES-1 cells,creating the PLGC cell model(MC cells).The MC cells were divided into the following groups for intervention with the water extract of the Jianpi Huoxue Decoction and folic acid solution:Group 1:GES-1+MNNG(MC cells);Group 2:GES-1+MNNG+low concentration(L)of the Decoction;Group 3:GES-1+MNNG+medium concentration(M)of the Decoction;Group 4:GES-1+ MNNG+high concentration(H)of the Decoction;Group 5:GES-1+MNNG+folic acid.The intervention effects of different concentrations of the Decoction on the viability,migration,and invasive ability of MC cells were assessed using the MTT assay,cell scratch experiment,and Transwell invasion assay,respectively.The morphological changes of MC cells were observed under a light microscope.2.2 Transcriptomic study of Jianpi Huoxue Decoction in MC cellsThe Jianpi Huoxue Decoction was used to intervene in MC cells,and RNA extraction,purification,library construction,and quality control were performed on the samples.Whole transcriptome sequencing was conducted to detect the differential expression of mRNAs and lncRNAs in MC cells before and after intervention with the Jianpi Huoxue Decoction.Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed to explore the signaling pathways possibly regulated by the Jianpi Huoxue Decoction,providing a basis for subsequent cellular experiments.2.3 Mechanism study of the Jianpi Huoxue Decoction in inhibiting EMT of MC cells through the TGF-β1/Smad3/CTGF signaling axis2.3.1 Effects of TGF-β1 agonists on MC cell growth,migration and invasion and the intervention effect of Jianpi Huoxue DecoctionThe intervention effects of the Jianpi Huoxue Decoction on the viability,migration,and invasive ability of MC cells stimulated by TGF-β1 agonist were observed using the MTT assay,cell scratch experiment,and Transwell invasion assay,respectively.The morphological changes of MC cells after stimulation with TGF-β1 agonist and intervention with the Jianpi Huoxue Decoction were observed under a light microscope.The specific groupings were as follows:Group 1:GES-1 MNNG(MC cells);Group 2:GES-1+MNNG+high concentration(H)of the Decoction;Group 3:GES-1+MNNG+TGF-β1 agonist;Group 4:GES-1+MNNG+TGF-β1 agonist+high concentration(H)of the Decoction.2.3.2 EMT inhibition of TGF-β1/Smad3/CTGF signaling axis and MC cells by the Jianpi huoxue DecoctionWestern Blot was performed to detect the effects of Jianpi Huoxue Decoction on the MC cells’ protein expression of TGF-β1,Smad3,CTGF,E-cadherin and Vimentin,and to detect the expression of each protein in MC cells before and after the intervention of Jianpi Huoxue Decoction after TGF-β1 agonist stimulation.Detailed grouping:Group 1:GES-1+MNNG(MC cells);Group 2:GES-1+MNNG+Jianpi Huoxue Decoction low concentration(L);Group 3:GES-1+MNNG+Jianpi Huoxue Decoction medium concentration(M);Group 4:GES-1+MNNG+Jianpi Huoxue Decoction high concentration(H);Group 5:GES-1+MNNG+folic acid;Group 6:GES-1+MNNG+TGF-β1 agonist;Group 7:GES-1+MNNG+TGF-β1 agonist+Jianpi Huoxue Decoction High Concentration(H).Results:1.Clinical Study1.1 Comparison of clinical efficacyThe total effective rate of the treatment group was 83.72%and the total effective rate of the control group was 58.54%,and the treatment group was higher than the control group,the comparison between the two groups was statistically significant(P<0.05).1.2 Comparison of traditional Chinese medicine syndrome efficacyThe total effective rate of the treatment group was 88.37%and the total effective rate of the control group was 60.98%,and the treatment group was higher than the control group,the comparison between the two groups was statistically significant(P<0.05).1.3 Comparison of traditional Chinese medicine syndrome scores:The TCM syndrome total score decreased in both groups after treatment,which was statistically significant(P<0.05),and the comparison between the two groups of TCM syndrome total score after treatment was lower in the treatment group than in the control group,which was statistically significant(P<0.05).In terms of the TCM syndrome individual scores,the scores of dull stomachache,gastric fullness,anorexia,thin stool,tiredness and fatigue,shortness of breath and no desire to talk after eating decreased in both groups after treatment(P<0.05),except for thin stools,all other TCM syndrome individual scores were lower in the experimental group than in the control group after treatment,which were statistically significant(P<0.05).There was no statistically significant comparison between the two groups after the treatment of thin stools score(P>0.05).1.4 Efficacy under gastric endoscopy and histopathologyThe gastroscopic gastric mucosal score decreased after treatment in both groups,which was statistically significant(P<0.05),and the comparison between groups of gastric mucosal score after treatment in both groups was lower in the treatment group than in the control group,which was statistically significant(P<0.05).In terms of histopathological efficacy,the gastric mucosal atrophy,intestinal epithelial hyperplasia,active and chronic inflammation scores decreased in both groups after treatment(P<0.05),and the treatment group was lower than the control group in the intergroup comparison of all scores after treatment in both groups,all of which were statistically significant(P<0.05).In terms of dysplasia,there was an improvement in both groups after treatment,yet there was no statistical significance(P>0.05).2.Experimental study2.1 In vitro inhibitory effects of the Jianpi Huoxue Decoction on the growth,migration,and invasion of MC cellsMTT assay showed that compared to the blank group,the viability of GES-1 cells significantly decreased after intervention with various concentrations of MNNG,and this decrease was statistically significant(P<0.05).The viability of GES-1 cells gradually decreased with increasing concentrations of MNNG,showing a concentration-dependent and time-dependent pattern.At an MNNG concentration of 40μmol/L,the inhibition rate on GES-1 cells was approximately 20%.Based on the references and preliminary experiments,the subsequent experiments were conducted with an MNNG intervention concentration of 40μmol/L and a duration of 48 hours.;Compared with the blank control group,the activity of GES-1 cells significantly increased after intervention with different concentrations of the Jianpi Huoxue Decoction and folic acid solution(P<0.05).Furthermore,the GES-1 cell activity gradually increased with increasing concentrations,showing concentration-dependence and time-dependence.The subsequent experimental intervention was set as follows:low,medium,and high concentrations of the Jianpi Huoxue Decoction were 4,8,and 16 mg/mL,respectively,with an intervention time of 48 hours.The intervention concentration of folic acid solution was 1 μg/mL,with an intervention time of 48 hours.Normal GES-1 cells grew attached to the wall of the culture dish,exhibiting spindle-shaped morphology and regular shape.After adding MNNG,some cells died,indicating the cytotoxicity of MNNG.With increasing intervention time,the surviving cells gradually exhibited irregular morphology,polygonal shape,and grew in clusters,showing tight and disordered arrangement resembling "islands." Vacuoles were observed in the cytoplasm,indicating successful MC cell modeling.Compared with the GES-1+MNNG group(MC cells),the morphology of MC cells improved after intervention with various concentrations of the Jianpi Huoxue Decoction and folic acid solution Peripheral protrusions and polygonal shapes decreased,arrangement became more regular,and the number of dead cells gradually decreased.Moreover,cell activity,cell migration rate,and invasive ability significantly decreased with statistical significance(P<0.05).Compared with the GES-1+MNNG+folic acid group,high concentration of the Jianpi Huoxue Decoction intervention resulted in a significant decrease in MC cell activity,migration rate,and invasiveness,with statistical significance(P<0.05).2.2 Transcriptional Study of the Jianpi Huoxue Decoction in MC cellsA total of 324 differentially expressed mRNAs were detected in MC cells after intervention with the Jianpi Huoxue Decoction.Among them,172 mRNAs were upregulated,and 152 mRNAs were downregulated.Additionally,a total of 845 differentially expressed lncRNAs were detected,with 468 lncRNAs upregulated and 377 lncRNAs downregulated.By KEGG enrichment analysis,the differentially expressed mRNAs revealed enrichment in signaling pathways closely associated with the "inflammation-cancer" transformation of chronic atrophic gastritis(CAG),including the PPAR signaling pathway,ErbB signaling pathway,Wnt signaling pathway,IL-17 signaling pathway,EGFR signaling pathway,JAK-STAT signaling pathway,TNF signaling pathway,PI3K-Akt signaling pathway,and TGF-β signaling pathway.In the TGF-β1 signaling pathway,ENSG00000125378 was upregulated,while ENSG00000136997,ENSG00000163235,and ENSG00000281453 were downregulated.2.3 Mechanism Study of the Jianpi Huoxue Decoction in Inhibiting EMT of MC cells through the TGF-β1/Smad3/CTGF Signaling Axis2.3.1 Effects of TGF-β1 agonist on the growth,migration,and invasion of MC cells and the intervention of the Jianpi Huoxue DecoctionCompared to the GES-1+MNNG group(MC cells),the morphology of MC cells became more irregular,with enlarged cell size,polygonal shape,disordered arrangement,increased clustered growth resembling "islands," and significantly increased cell viability,migration,and invasion after TGF-β1 agonist intervention,which was statistically significant(P<0.05).Compared to the GES-1+MNNGH+TGF-β1 agonist group,MC cells stimulated by TGF-β1 agonist and intervened with the Jianpi Huoxue Decoction showed improved morphology,reduced peripheral protrusions and polygonal shape,more regular arrangement,decreased number of dead cells,orderly arrangement,decreased clustered growth resembling "islands,"and significantly decreased cell viability,migration,and invasion,which was statistically significant(P<0.05).2.3.2 Inhibitory Effects of the Jianpi Huoxue Decoction on the TGF-β1/Smad3/CTGF Signaling Axis and EMT in MC cellsWestern blot analysis revealed that compared to the GES-1+MNNG group(MC cells),the expression levels of TGF-β1,Smad3,CTGF,and Vimentin in MC cells significantly decreased,while the level of E-cadherin significantly increased after intervention with various concentrations of the Jianpi Huoxue Decoction and folic acid solution(P<0.05).Compared to the GES-1+MNNG+folic acid group,high concentration of the Jianpi Huoxue Decoction intervention significantly decreased the protein expression levels of TGF-β1,Smad3,CTGF,and Vimentin in MC cells,while significantly increasing the protein expression level of E-cadherin(P<0.05).Compared to the GES-1+MNNG group(MC cells),intervention with TGF-β1 agonist significantly increased the protein expression levels of TGF-β1,Smad3,CTGF,and Vimentin in MC cells(P<0.05),while significantly decreasing the protein expression level of E-cadherin with statistical significance(P<0.05).Compared to the GES-1+MNNG+TGF-β1 agonist group,high concentration of the Jianpi Huoxue Decoction intervention after TGF-β1 agonist stimulation significantly decreased the protein expression levels of TGF-β1,Smad3,CTGF,and Vimentin(P<0.05),while significantly increasing the protein expression level of E-cadherin(P<0.05),with statistical significance.Conclusion:1.The Jianpi Huoxue Decoction can significantly alleviate the clinical symptoms of patients with pre-cancerous lesions of CAG with spleen deficiency and blood stasis,improve patients’gastroscopic gastric mucosal signs,reverse gastric mucosal atrophy and intestinal epithelial hyperplasia,and also have some inhibitory effects on heterotypic hyperplasia.2.Whole-transcriptome sequencing showed that the signaling pathway enriched by the differential mRNAs of MC cells in the PLGC cell model after the intervention of Jianpi Huoxue Decoction was closely related to the "inflammation-cancer" transformation of CAG,and Jianpi Huoxue Decoction may play a therapeutic role in this disease by regulating the TGF-β1 signaling pathway,which provides a direction for the research of mechanism of action of Jianpi Huoxue Decoction.3.By suppressing the TGF-β1/Smad3/CTGF signaling axis,Jianpi Huoxue Decoction inhibit the EMT process in MC cells and thereby preventing the "inflammation-cancer" transformation.This might possibly be one of the mechanisms in treating CAG precancerous lesions.