Jianpi Huayu Jiedu Recipe Through DNA Damage Repair Pathway Study On The Mechanism Of Treating Precancerous Lesions Of Gastric Cancer

ObjectiveJianpi Huayu Jiedu Formula can regulate the HR signaling pathway,repair DNA damage mediated by DSBs damage,and then reverse the progression of gastric precancerous lesions.Based on the above research hypotheses and content,this study aims to achieve the following1.To explore the molecular mechanism of malignant progression of precancerous lesions caused by inflammatory damage of gastric mucosa induced by DSBs,and provide new ideas for revealing the pathogenesis of GPL;2.Based on the pharmacological research of traditional Chinese medicine related to strengthening the spleen,resolving blood stasis,and detoxifying,it is clarified that the Jianpi Huayu Jiedu Formula regulates the HR signaling pathway to repair DSBs damage,and exerts the preventive and therapeutic effects of traditional Chinese medicine on gastric precancerous lesions;3.Search for candidate genes related to gastric cancer and gastric precancerous lesions through a database to find new targets for the prevention and treatment of gastric cancer and gastric precancerous lesions.Methods1.In vivo experiment,Jianpi Huayu Jiedu Recipe intervened in MNNG induced DNA damage mice of gastric precancerous lesions,observed the pathological changes of gastric mucosa,inflammatory factors,ultrastructural changes,DNA damage,and activation of DNA damage repair pathway,and explored the pathological mechanism of Jianpi Huayu Jiedu Recipe to repair DNA damage and improve the malignant progress of gastric mucosa;2.In vitro experiments,construct a cell damage model of DSBs and treat it with the water extract of Jianpi Huayu Jiedu Formula.Detect the damage,cell cycle arrest,and cell proliferation of DSBs,and elucidate the molecular mechanism of Jianpi Huayu Jiedu Formula in promoting DNA damage repair and preventing precancerous lesions of gastric cancer3.Using TIMER database to comprehensively analyze the expression of ABCA1 m RNA and protein in tumor tissue and corresponding normal tissues adjacent to cancer;Using the Clinical Proteomics Tumor Analysis Collaborative Group(CPTAC)database to analyze the differential expression of ABCA1 protein between tumor tissue and normal tissue;Using the Gene Expression Profile Interaction Analysis website(GEPIA)to analyze the relationship between ABCA1 expression and gastric cancer staging;Kaplan Meier Plotter online web analytics was used to analyze the relationship between ABCA1 expression and survival and prognosis of gastric cancer patients;The TIMER database was used to analyze the correlation between ABCA1 protein expression and tumor immune infiltrating cells in gastric cancer tissue.Results1.A mouse model of gastric precancerous lesions was replicated by using the Jianpi Huayu Jiedu formula to pre prepare BALB/c mice and MNNG for free drinking.Compared with the model group,the intervention of Jianpi Huayu Jiedu Formula and positive drugs has varying degrees of impact on weight changes,food and water intake,spleen index,and thymus index;And reduce the inflammatory factor IL-1 to varying degrees β、 IL-6、TNF-α m RNA level.The pathological changes observed by HE staining showed that,compared with the blank group,the glands in the lamina propria of the gastric mucosa in the model group were atrophied and disordered,intestinal metaplasia appeared in the mucosa base and lamina propria,and vacuolar cells were visible.The nuclei near the mucosa base were deeply dyed purple or purple black,and the cytoplasm was blue purple.After the intervention of Jianpi Huayu Jiedu Recipe,the arrangement of glands was more regular than that of the model control group.There were new glands in the muscularis mucosae and lamina propria,with normal color and morphology.2.The ultrastructural changes of the mouse gastric mucosa epithelium in each group were observed under the transmission electron microscope.The glandular epithelial cells in the blank group were closely arranged,with oval nuclei,obvious perinuclear spaces,rich euchromatin in the nucleus,and clear nucleoli;Mitochondria and rough endoplasmic reticulum are abundant in the cytoplasm.Mitochondria in the cytoplasm are large and dense,oval,steep,dense and clear.A large number of round secretory granules are distributed at the base of the cytoplasm.The granules have two types,high electron density and medium electron density.The basal membrane of epithelial cells in the model group is tortuous and uneven,with few secretory particles in the cytoplasm;The mitochondrial matrix is blurry and the cristae are unclear;The rough endoplasmic reticulum is rich and autophagosomes appear.Compared with the model group,the nuclear membrane of each dose group of Jianpi Huayu Jiedu Recipe and the positive drug group was regular round,and heterochromatin was reduced;The swelling and expansion of rough endoplasmic reticulum were reduced;The number of mitochondria increases,with intact morphology and dense cristae.3.Using Western blot technology to detect the expression levels of γ-h2 ax which is DNA damage marker proteins and DNA damage repair related pathway proteins p-ATM,ATM,pChk2,and Chk2.Discovered in the model control group γ-The expression level of H2 ax was significantly increased compared to the blank control group mice(P<0.01).Under the treatment of Jianpi Huayu Jiedu Formula and positive drugs,its relative expression level decreased to varying degrees.Among them,the low-dose group of Jianpi Huayu Jiedu Formula and the high-dose group of Jianpi Huayu Jiedu Formula were compared to the reduced model control group γ-The level of H2 ax protein was statistically significant(P<0.05).Compared with the blank control group,the protein expression levels of pATM/ATM(P<0.001)and p-Chk2/Chk2(P<0.001)in the model control group were significantly increased;The expression levels of p-ATM/ATM and p-Chk2/Chk2 proteins decreased after the intervention of Jianpi Huayu Jiedu Formula and positive drugs.The above results indicate that the improvement of MNNG induced DNA damage by Jianpi Huayu Jiedu Formula may be related to the regulation of ATM/Chk2 pathway.The expression levels of key DNA damage repair proteins RAD51(P<0.05)and RAD52(P<0.01)in the HR signaling pathway model control group were significantly reduced compared to the blank control group mice.After the intervention of Jianpi Huayu Jiedu Formula and positive drugs,their relative expression levels increased to varying degrees.The immunohistochemical results showed that the key protein of the HR signaling pathway,BRCA1,was almost entirely expressed in the cytoplasm of the model control group compared to the blank control group,with a lower staining intensity;Compared with the model control group,the expression intensity of BRCA1 protein increased after the intervention of Jianpi Huayu Jiedu Formula and positive drugs.4.The use of MNNG to induce DNA damage in GES-1 cells has a dose-dependent effect on MNNG concentration.MTT and comet assay results indicate that as MNNG concentration increases,DNA damage in GES-1 cells becomes more severe.Jianpi Huayu Jiedu Formula exerts different regulatory effects on DNA damage induced by different concentrations of MNNG,with MNNG modeling concentration of 5 μmol,the cell survival rate of each dose group of Jianpi Huayu Jiedu Formula increased with the increase of drug concentration.Compared with the model group,JPHYJD5 μg/ml,the cell survival rate was statistically significant(P<0.01)JPHYJD10 μ The survival rate of g/ml cells significantly increased(P<0.001).When MNNG modeling concentration is 10 μmol,the cell survival rate of each concentration group of Jianpi Huayu Jiedu Formula decreased with increasing drug concentration.Compared with the model group,JPHYJD5,10 μg/ml,the cell survival rate was statistically significant(P<0.01).Flow cytometry was used to detect the change of cell cycle proportion.Compared with the blank control group,the proportion of cells in G0/G1 phase of GES-1 cells was significantly reduced after MNNG induction,and the proportion of cells in S phase was increased.After JPHYJD intervention,the proportion of G2/M cells was increased or decreased,and there was no obvious dose-dependent effect.The above results suggest that JPHYJD can inhibit the transformation from S phase to G2 phase phase and induce S/G2 phase arrest under the induction of two MNNG concentrations.5.TIMER database analysis showed that compared with normal tissues adjacent to cancer,the expression level of ABCA1 m RNA in gastric cancer(STAD)and esophageal cancer(ESCA)tissues was significantly increased(P<0.01),while the expression level of ABCA1 m RNA in breast infiltrating cancer(BRCA),cholangiocarcinoma(CHOL),colon cancer(COAD),rectal adenocarcinoma(READ)tissues was significantly reduced(P<0.01)01);Analysis of the CPTAC database showed that the expression level of ABCA1 in gastric cancer tissue was higher than that in normal tissue and was associated with poor prognosis of gastric cancer(P<0.05);And it is related to clinical characteristics such as race,age,and pathological stage(P<005);Single and multiple factor Cox regression analysis showed that ABCA1 m RNA expression,age of onset,and TNM stage were independent risk factors affecting the prognosis of gastric cancer(P<0.05);SSGSEA analysis showed that the expression of ABCA1 was significantly correlated with the infiltration level of natural killer cell,macrophages,immature dendritic cells(i DCs),and effector memory T cell(Tem)(P<0.001).Through molecular docking,it was found that the effective active ingredients of Jianpi Huayu Jiedu Formula have a good binding force with the ABCA1 gene,and experimental verification found that Jianpi Huayu Jiedu Formula can reduce the ABCA1 m RNA level in gastric cancer cells.Conclusion1.MNNG mainly induces DNA damage in cells and promotes the production of inflammatory factors,ultimately leading to pathological changes in gastric precancerous lesions.The Jianpi Huayu Jiedu formula can activate the HR signaling pathway,upregulate the key HR proteins BRCA1,Rad51,and Rad52,inhibit the ATM/Chk2 axis,and restore normal mitosis to promote DNA repair.2.Jianpi Huayu Jiedu Recipe can cause cell cycle arrest in S/G2 phase to DNA damage induced by different concentrations of MNNG,and has a two-way regulatory effect on the proliferation and apoptosis induction of GES-1 cells.The degree of effect is positively related to the drug concentration,and its mechanism may be related to the degree of DNA damage.However,further in-depth research is needed on how it regulates cell proliferation or apoptosis.3.Jianpi Huayu Jiedu Recipe can improve the overall situation of mice with precancerous lesions,reduce inflammatory factors,promote the recovery of pathological structure and ultrastructure of gastric mucosa epithelium,maintain normal physiological functions,and play a role in preventing and treating precancerous lesions.4.ABCA1 is upregulated in gastric cancer,and its high expression is associated with poor prognosis in gastric cancer patients.It can become a promising diagnostic indicator,treatment target,and prognostic marker for gastric cancer.The Jianpi Huayu Jiedu formula can assist in the treatment of gastric cancer by reducing the expression of ABCA1.