| Objective:(1)To explore and summarize the clinical experience of Professor Lyu Wenliang using Lianpuyin Modified and Subtracted Decoction to block the"inflammatory cancer transformation"of chronic gastritis;(2)To explore the core target and network mechanism of Lianpuyin Modified Decoction in the treatment of chronic atrophic gastritis based on network pharmacology;(3)To explore the regulatory effect of Lianpuyin Modified Decoction on inflammatory microenvironment and core target in angiogenesis of chronic atrophic gastritis.Methods:(1)By means of literature research,the pathological mechanism of chronic gastritis"transformation of inflammatory cancer"and the prevention and treatment theory of traditional Chinese medicine were systematically analyzed and discussed by collecting and referring to domestic and foreign literatures about chronic gastritis"transformation of inflammatory cancer".(2)Qualitative research method was adopted,and the grounded theory was used to make the interview outline.Professor Lyu Wenliang was interviewed in depth,and the interview data were transcribed.The collected data were combined with the observation method and literature review.To systematically analyze and summarize Professor Lyu Wenliang's main thinking mode of clinical disease differentiation and syndrome differentiation,his understanding of the etiology and pathogenesis of chronic gastritis"transformation of inflammation and cancer",his understanding of the syndrome of Lianpuyin and his diagnosis and treatment experience of blocking the transformation of inflammation and cancer with Lianpuyin plus or subtracted formula,and to extract his academic thoughts.(3)Based on the network pharmacological research method,the targets of Lianpuyin additive and subtracted formula for TCM and chronic atrophic gastritis were respectively retrieved from Symmap and Disgenet database.The common targets of the two were used as potential therapeutic targets,and introduced into the String 11.0analysis platform for PPI analysis.The target network with the lowest correlation coefficient greater than 0.9 was calculated and analyzed with Cytoscape3.7.2 software,and the top ten targets with the highest acquisition value were taken as core action targets.The common targets were imported into the David 6.8 analysis platform for GO and KEGG enrichment analysis.CC,BP and MF items with P value less than 10-5and the number of targets greater than 5 were screened for cluster analysis.The signal transduction pathway most closely associated with the pathological mechanism of chronic atrophic gastritis was screened for mechanism prediction.(4)A total of 62 male SPF SD rats were randomly divided into normal control group(group A)(n=10)and model group(n=52).The model group was replicated by multi-factor compound method.The free drinking amount of 120mg·L-1MNNG solution was given daily,and1ml/100g MNNG solution was gavaged twice a week(week 1 and week4).Fasting once 2 days after eating,and feeding with ranitidine containing 0.03g·kg-1during feeding time;On the first day after eating,15%Na Cl was gavaged at 56?,1ml/100g for each animal;On the day of fasting,40%ethanol was given by gavage,1ml/100g for each animal.After 8 and 14 weeks of continuous modeling,one rat in each group was killed randomly,and the gastric tissue was extracted and routine HE staining was performed.Microscopic observation confirmed the atrophy of intrinsic glands in the gastric mucosa,indicating the success of modeling.During the modeling period,5 rats in the model group died.After successful modeling,the rats in model group were randomly divided into model group(group B),vitrease group(group C),coptis magnolia magnolia group(group D),Lianpuyin Modified Decoction formula group(group E)and Lianpuyin group(group F),with 9 rats in each group.Group C,D,E and F were treated with vitasein,coptis chinensis Magnolia,Lianpuyin Modified Decoction formula and Lianpuyin,respectively.The gavage dose was converted according to the daily dose of adult body weight(60kg),and then the rats were given gavage.During the intervention of traditional Chinese medicine,1 animal died in group D and 2 died in group F.After the intervention,the rats were anesthetized with chloral hydrate(0.35ml/100g),and the blood of the abdominal aorta was extracted.After centrifugation(3000rpm,10min),the serum was collected,and the contents of IL-1?,IL-6,IL-17 and TNF-?in the serum were determined by ELISA.Some gastric tissues were collected and cryopreserved at-80?.The expressions of STAT3 m RNA,JAK2 m RNA and HIF-1A m RNA in gastric tissues were detected by RT-PCR,and the relative expression levels of p-JAK2 and p-STAT3 were detected by WB method.The other part of the gastric tissue was fixed with 10%formaldehyde solution,and the morphological changes of gastric tissue were observed by HE staining,and the expression of EGFR and VEGF in gastric tissue was detected by immunohistochemical method.Results:(1)Through qualitative research,more than 160 published academic papers,8 special reports,5 audio and video lectures,8notes with teachers and 2 monographs by Professor Lyu Wenliang;A total of 12 interviews were conducted,and 4 thematic threads were extracted after data transcription.(2)A total of 367 drug targets,203 disease targets and 40common targets were obtained from the database.PPI analysis showed that STAT3,VEGFA,IL6,CXCL8,TNF,IL1B,IL10,TP53,EGFR and AKT1were the core targets.Two clusters were obtained by GO enrichment,CC was mainly involved in extracellular regions,BP was mainly involved in inflammatory response,immune response and positive regulation mediated by interleukin-6,and MF was mainly involved in cytokine activity.Thirty entries were obtained by KEGG enrichment,and the closely related signaling pathways were PI3K-Akt,HIF-1,MAPK,Fox O and so on.(3)Experimental results:(1)Morphological observation showed that after 14 weeks of modeling,the gastric tissue of rats was dark,wrinkled and flat,and part of the tissue was wart-like.After 8weeks of experimental intervention,the gastric mucosa tissues of rats in group A were light red and bright,with smooth surface,regular folds and A small amount of mucus.Gastric mucosal tissues of rats in group B were dark in color,with low and flat folds,thin in thickness and wart-like eminence on the surface.In each treatment group,the gastric tissue was dark red,regular,and a small amount of verrucous eminence on the surface.By HE staining,microscopic observation showed that the gastric tissue of rats in group A had complete epithelium,regular arrangement of cells,dense arrangement of glands,and no intestinal metaplasia and inflammation.In group B,there was a large amount of inflammatory infiltration in gastric mucosa,with less atrophy of glands and irregular arrangement,and epithelial intestinal metaplasia.In groups C and D,the degree of inflammatory cell infiltration and atrophy was reduced,and intestinal metaplasia was still visible.Group E and F had mild inflammatory infiltration,while group E was milder than group F.(2)ELISA showed that the contents of IL-1?,IL-6,IL-17 and TNF-?in serum of group B were significantly higher than those of group A(P<0.05);Groups D,E and F were significantly lower than group B(P<0.05);The levels of IL-6,IL-17 and TNF-?in group C were significantly decreased compared with group B(P<0.05),and those in groups E and F were significantly higher than those in group C(P<0.05).The level of IL-17 in group F was better than that in group D(P<0.05),and that in group E was better than that in group F(P<0.05).The level of TNF-?in groups E and F was significantly higher than that in group D(P<0.05).(3)The expression of EGFR and VEGF in group B was significantly higher than that in group A(P<0.05),and the expression of EGFR and VEGF in group E and F was significantly lower than that in group B(P<0.05),and that in group E was significantly lower than that in group D and F(P<0.05).EGFR expression in groups C and D was significantly lower than that in group B(P<0.05),and that in group E was significantly lower than that in group C(P<0.05).The expression of VEGF in groups E and F was significantly lower than that in group C(P<0.05).(4)RT-PCR showed that the levels of STAT3 m RNA,JAK2 m RNA and HIF-1A m RNA in group B were significantly higher than those in group A,and those in group E were significantly lower than those in groups B,C and D(P<0.05).The m RNA level of STAT3 in groups D and F was significantly decreased compared with group B(P<0.05),and that in group F was significantly decreased compared with group C and D(P<0.05).The m RNA level of JAK2 in group E was significantly lower than that in group F(P<0.05).The m RNA level of HIF-1A in group F was significantly decreased compared with that in groups B and C(P<0.05).(5)WB experiment showed that the relative expression levels of p-STAT3 and p-JAK2 in group B were significantly higher than those in group A(P<0.05);The relative expression level of p-STAT3in group E was significantly lower than that in group B(P<0.05).The relative expression level of p-JAK2 in groups C,D,E and F was significantly lower than that in group B(P<0.05).Conclusion:(1)Lianpu Yin is a common prescription used by Professor Lv Wenliang to treat dampness-heat syndrome of spleen and stomach of chronic gastritis.Under the thinking mode of combining disease and syndrome and corresponding prescription and syndrome,combined with the characteristics of disease and syndrome in the process of"transformation of inflammation and cancer"of chronic gastritis,it is concluded that:"Dampness and heat depress evil"and"Dampness and heat cause blood stasis"are the key etiology and pathogenesis in the process of pathological evolution.Based on the syndrome of Lianpuyin,on the basis of reducing dampness and clearing heat,the addition and subtraction prescription of Lianpuyin,which is supplemented with drugs to promote blood circulation and remove blood stasis,is suitable for the clinical treatment of chronic atrophic gastritis with remarkable curative effect.(2)Lianpuyin can improve the inflammatory microenvironment and repair the morphology of gastric mucosa by down-regulating the expressions of serum inflammatory cytokines IL-1?,IL-6,IL-17 and TNF-?,and inhibiting the expressions of STAT3 m RNA and JAK2 m RNA and the levels of p-JAK2 and p-STAT3 in gastric tissues.By down-regulating EGFR,VEGF and HIF-1A m RNA expression,inhibit angiogenesis,and then play a therapeutic role in chronic atrophic gastritis.The mechanism of action may also involve multiple targets in multiple biological pathways. |
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