| Objective:This study uses high-throughput mRNA sequencing technology to screen the signal pathways and core pathogenic genes closely related to the pathogenesis of Gastric Ulcer(GU)spleen stomach deficiency cold syndrome,and explore the intervention effect and therapeutic mechanism of Lizhong Decoction on GU spleen stomach deficiency cold syndrome through animal experiments.Method:1.Twenty-four male SD rats were randomly divided into normal group and model group,with 12 rats in each group.A combined model of GU spleen stomach deficiency and cold syndrome was established.High throughput mRNA sequencing technology was used to sequence normal rats and GU rats with spleen stomach deficiency and cold syndrome.The differentially expressed genes obtained by sequencing matching were analyzed using edgeR packages.The Metascape platform was used to perform GO function analysis and KEGG pathway enrichment analysis on significantly differentially expressed genes.Screening for core pathogenic genes of GU spleen stomach deficiency cold syndrome was conducted on the STRING platform and Cytascape 3.7.1 software for genes enriched in the pathogenesis related pathways of GU spleen stomach deficiency cold syndrome.2.Sixty male SD rats were randomly divided into normal group,GU spleen stomach deficiency cold group,Esomeprazole(ESO)group,low dose Lizhong Decoction group(3.75 g/kg),and high dose Lizhong Decoction group(7.5 g/kg),with 12 rats in each group.After sampling,the ulcer index(UI)and ulcer inhibition rate(CI)of the rat gastric mucosa were measured by naked eye observation and photography;HE staining method was used to observe the morphological changes of gastric mucosa under microscope,and the pathological findings were scored under microscope;The content of serum malondialdehyde(MDA),reduced glutathione(GSH)and the activity of pepsin in gastric tissue were measured by ELISA;The expression of CASP3,VCAM1,MAPK15,and MMP3 genes in rat gastric tissue was measured by ddPCR;Molecular docking verifies the affinity between the active components in Lizhong Decoction and the target protein of GU spleen stomach deficiency cold syndrome;Immunofluorescence assay was used to detect the expression of CASP3,VCAM1,MAPK15,and MMP3 proteins in rat gastric tissue.Result:1.Screening of key pathogenic pathways and core genes in GU with spleen stomach deficiency and cold syndrome by high throughput mRNA sequencing We screened 1 791 differentially expressed genes between GU rats with spleen stomach deficiency and cold syndrome and normal rats,including 542 up-regulated genes and 1 249 down-regulated genes;There were 820 significantly differentially expressed genes,including 177 up-regulated genes and 643 down-regulated genes.Through GO enrichment analysis,1 077 biological process related items,104 cell component related items,and 197 molecular function related items were obtained.Enrichment analysis of KEGG signal pathway obtained 38 pathways associated with GU spleen stomach deficiency cold syndrome,such as IL-17 signal pathway,TNF signal pathway,etc.After expanding all 222 genes enriched in the KEGG pathway,a PPI network with 652 nodes and 4 119 edges was constructed.The network was analyzed using the Degree algorithm in Cytascape,and ten GU core pathogenic genes of spleen and stomach deficiency cold syndrome,including PTPRC,EGFR,CD8A,EGF,VCAM1,SYK,CASP3,CCL5,MAPK15,and MMP3,were obtained.2.Interventional effect and mechanism of Lizhong decoction on GU rats with spleen stomach deficiency and cold syndrome① Compared with the normal group rats,the UI of the GU spleen stomach deficiency cold group rats significantly increased(P<0.01).The UI of rats treated with ESO and Lizhong Decoction at low and high doses decreased significantly compared to GU group with spleen stomach deficiency and cold(P<0.05).The inhibitory rate of high dose Lizhong Decoction on GU was 86.95%.② The results of HE staining observation and pathological scoring showed that compared with the normal group,the gastric mucosal structure in the GU spleen stomach deficiency cold group was incomplete,with severe edema,exfoliation,and formation of pits in epithelial cells,disordered gland arrangement,and a large number of inflammatory cell infiltration in the submucosa(P<0.01).Compared with the GU spleen stomach deficiency cold group,the morphology of the gastric mucosa in each treatment group was similar to that of the normal rats,with most of the regions having complete structures,glands arranged neatly,no epithelial exfoliation was observed,inflammatory cell infiltration and edema were reduced,and the total pathological score was significantly decreased(P<0.05).③ The ELISA results showed that compared with the normal group,the serum MDA content and pepsin activity in the GU spleen stomach deficiency cold group were significantly increased(P<0.01);The content of GSH decreased significantly(P<0.01).After drug treatment,the serum MDA content and pepsin activity in the low-dose and high-dose ESO and Lizhong Decoction groups were significantly lower than those in the model group(P<0.01),while the GSH content was significantly higher(P<0.01).④ The results of ddPCR showed that compared with normal rats,the expression levels of CASP3 and VCAM1 genes in gastric mucosa of GU rats with spleen stomach deficiency and cold syndrome decreased(P<0.05),while the expression levels of MAPK15 and MMP3 genes increased(P<0.01).Compared with the model group,the CASP3 and VC AM1 gene contents in the ESO,low dose Lizhong Decoction group,and high dose Lizhong Decoction group were significantly increased(P<0.05),while the MAPK15 and MMP3 gene contents were significantly decreased(P<0.01).⑤ The results of molecular docking showed that the active components of Lizhong Decoction,syringin,glycyrrhizin,atractylolactone I,and 10-gingerol,had a good docking with the target proteins CASP3,VCAM1,MAPK15,and MMP3 of GU spleen stomach deficiency and cold syndrome.⑥ The results of immunofluorescence detection showed that compared with the normal group,the expression of CASP3 and VC AM1 proteins in the gastric mucosa of the model group was significantly decreased(P<0.01)while the expression of MAPK15 and MMP3 proteins were significantly increased(P<0.01).Compared with the model group,the expressions of CASP3 and VCAM1 proteins in the gastric mucosa of rats in the low and high dose Lizhong Decoction groups were significantly increased(P<0.01),while the expressions of MAPK15 and MMP3 proteins were significantly decreased(P<0.01).Conclusion:1.Based on high throughput mRNA sequencing technology,two signal pathways,IL-17 and TNF,were screened out,which are closely related to the pathogenesis of GU with spleen stomach deficiency and cold syndrome.Ten core pathogenic genes of GU with spleen stomach deficiency and cold syndrome,including PTPRC,EGFR,CD8A,EGF,VCAM1,SYK,CASP3,CCL5,MAPK15,and MMP3,were further identified.2.Lizhong Decoction can promote the healing of ulcers in rats with GU spleen stomach deficiency and cold syndrome,and its intervention mechanism may be related to upregulation of CASP3 and VCAM1 gene expression,downregulation of MAPK15 and MMP3 gene expression,inhibition of oxidative stress,and elimination of inflammation. |
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