| Background and objectives:As we all know,our country is a high incidence of gastric cancer,and Helicobacter pylori(H.pylori)infection is an important factor in its development.For early gastric cancer caused by H.pylori infection,we can usually use EMR(endoscopic mucosal resection,EMR)and other operations for therapy,but some patients still develop advanced gastric cancer due to persistent infection with H.pylori.At present,in the consensus on the prevention of gastric cancer,it is pointed out at home and abroad that the eradication of H.pylori is the key primary prevention measure.Although there are many treatment options for H.pylori eradication,some patients are still not successfully eradicated.Recent studies have found that H.pylori not only adheres to the surface of gastric mucosal cells,but also invades into the cells to further colonize and survive.The bacterial internalization of H.pylori can make it escape the antibacterial action of antibiotics,so as to avoid being inhibited or killed by antibiotics,and then once the conditions are suitable,it can come out of the cells again,colonize the gastric mucosal surface and multiply.In addition,relevant studies have also confirmed that,compared with successfully eradicated H.pylori,the internalization level of failed H.pylori is significantly higher,which suggests that the internalization of bacteria may be an important reason for the failure of eradication of H.pylori.It can be seen that finding effective therapeutic targets against the internalization of H.pylori will lay a theoretical foundation for the establishment of an efficient eradication program.Autophagy is a cellular process in which lysosomes are utilized to degrade cellular contents.It not only plays a fundamental role in normal physiological processes but also is widely involved in various pathological processes.In recent years,the relationship between autophagy and microbial infections has become a research hotspot.However,the role of autophagy in host resistance to microbial infections is not yet fully understood,and its function in the body’s clearance of H.pylori infection,an important mechanism for microbial defense and inflammation immune regulation,is also unclear.Based on current research on H.pylori and the autophagy-lysosome pathway,we believe that it is crucial to elucidate the molecular mechanisms by which H.pylori reversely regulates the autophagy-lysosome pathway and to find drugs that target the restoration of damaged autophagy-lysosome function in order to help the body clear H.pylori through its own defense system.Berberine is mainly used in the clinical treatment of intestinal infections.It has a broad spectrum of antibacterial activity,showing inhibitory effects against many gram bacteria such as Staphylococcus aureus,Streptococcus hemolyticus and Vibrio cholerae.It is not cross-resistant with penicillin and erythromycin.Currently,with the increasing prevalence of H.pylori resistance,the potential role of berberine in combating H.pylori is also concerned.Studies have confirmed that berberine has an anti-H.pylori effect in vitro(MIC50=12.5mg/ml).Furthermore,some studies suggest that adding berberine to the traditional triple therapy can improve H.pylori eradication rates and related clinical symptoms.However,the precise antibacterial mechanism wielded by berberine remains obscure,and no reports have been issued regarding whether it facilitates clearing of intracellular H.pylori.Drawing upon the aforementioned research background,considering the significant importance of the autophagy-lysosome pathway in defending against microbial invasion and the potential advantages of berberine in the field of antibacterial research in recent years,we propose the hypothesis that berberine may remove intracellular H.pylori by targeting the activation and repair of the autophagy-lysosome pathway.This research initiative endeavors to investigate these matters and provide theoretical basis for the development of novel strategies aimed at eradicating H.pylori.Materials and Methods:I.To verify the effect of berberine removing H.pylori in vivo and in vitro1.The effect of berberine removing intracellular H.pylori: By constructing an H.pylori-infected HFE145 cell model,the clearing effect of berberine on intracellular H. pylori was observed.The specific 16 S r DNA content of H.pylori in cells was detected using q RT-PCR,and the localization and expression of H.pylori were observed using immunofluorescence.The colony-forming unit(CFU)experiment was used to count the number of H.pylori bacteria.2.The effect of berberine removing H.pylori in mice: By constructing an H.pylori-infected C57BL/6 mouse model,the clearing effect of berberine on H.pylori in the mouse body was observed.The specific 16 S r DNA content of H.pylori in cells was detected using q RT-PCR,and the localization and expression of H.pylori were observed using immunofluorescence.The CFU experiment was used to count the number of H.pylori bacteria,and further pathological changes in mouse gastric tissue were observed using qRT-PCR and H&E staining.3.The effect of berberine removing clinical antibiotic-resistant strains of H.pylori in human gastric epithelial cells: By constructing an H.pylori clinical antibiotic-resistant strain(532 and 536)infected HFE145 cell model,the clearing effect of berberine on the clinical antibiotic-resistant strains of H.pylori was observed.The specific 16 S r DNA content of H.pylori in cells was detected using q RT-PCR.II.Clarification that berberine removing H.pylori through targeting activation and repair of the autophagy-lysosome pathway1.Confirming the activation of autophagy-lysosome pathway by berberine: Using transmission electron microscopy to observe the effect of berberine on the ultrastructure of autophagy-related structures in H.pylori-infected cells.2.Confirming the H.pylori clearance by berberine through autophagy-lysosome pathway: Inhibiting key nodes of autophagic flux(using autophagy inhibitors 3-MA,WM,and Baf-A1)to observe the ability of berberine to clear H.pylori in cells,knocking down the expression of autophagy-related genes(using si ATG5 and si BECN1)to observe the ability of berberine to clear H.pylori in cells,and detecting the level of H.pylori-specific 16 S r DNA by q RT-PCR.3.Confirming the ability of berberine to repair the damaged autophagy-lysosome pathway in H.pylori infection: Using Western blot to detect the expression of autophagy-lysosome proteins in H.pylori-infected cells and H.pylori-infected mice after treatment with berberine,and further using Western blot to detect the expression of autophagy-lysosome proteins in H.pylori-infected cells after inhibiting autophagic flux formation.III.Exploring related molecules that can mediate the removal of H.pylori by berberine through the autophagy-lysosome pathway1.Verification in cell models: Through a review of recent literature related to berberine,molecules that may mediate the activation and repair of the autophagy-lysosome pathway by berberine are selected.Gene silencing technology is used to knock down the expression of the above molecules in cells,and then q RT-PCR is used to detect changes in the ability of berberine to clear H.pylori in gene-knockdown cells.Western blot is also used to detect changes in the expression levels of relevant autophagy-lysosome proteins in gene-knockdown cells regulated by berberine.2.Verification in gene-knockout mouse models: By constructing a gene-knockout mouse model,the effect of berberine on H.pylori clearance in vivo through this molecule is observed.q RT-PCR,immunofluorescence,and CFU experiments are used to observe changes in the ability of berberine to clear H.pylori in gene-knockout mice,and Western blot is used to further detect changes in the expression levels of relevant autophagy-lysosome proteins in gene-knockout mice regulated by berberine.IV.Investigating and validating key downstream target genes activated and repaired by berberine1.Finding differentially expressed autophagy-related genes via transcriptome sequencing: First,integrating the data from each group,and the establishing an overlapping gene set related to autophagy to further narrow down the range of downstream target genes and find differentially expressed autophagy-related genes.2.Screening for key downstream target genes activated by berberine for autophagy-lysosome pathway repair: Key downstream target genes are screened through multiple in vivo and in vitro experiments,and their protein level changes are confirmed by Western blot to be consistent with changes in transcription level.3.Validating key downstream target genes: Firstly,Western blot was used to detect the protein expression level of key downstream target genes in NLRP3 knockout cells and mouse models.Then si RNA was further used to silence the expression of key downstream target genes,q RT-PCR was used to detect the effect of berberine removal of intracellular H.pylori,and Western blot was used to detect the effect of berberine repair related autophagic lysosomal protein levels.V.Elucidating the specific mechanism of berberine regulation of key downstream target genes1.Predicting the transcription factors that may activate key downstream target genes: The promoter sequences of key downstream target genes are extracted from the NCBI database,and then transcription factors that may activate these genes are identified using databases such as JASPAR and PROMO.In addition,protein-protein interaction networks are predicted using the STRING database to further predict the potential transcription factors involved.2.Verifying the specific regulatory mechanism of transcription factors in the upstream-downstream relationship: Immunoprecipitation(Co-IP),Western blot,immunofluorescence,chromatin immunoprecipitation(Ch IP)and gene silencing were used to further elucidate the specific regulatory mechanisms of transcription factors in upstream and downstream relationships.Results:I.To verify the effect of berberine removing H.pylori in vivo and in vitro1.Berberine effectively removes intracellular H.pylori: In the H.pylori-infected HFE145 cell model,q RT-PCR,immunofluorescence,and CFU results show that berberine can significantly reduce the content of H.pylori in cells.2.Berberine effectively removes H.pylori in mice: In the H.pylori-infected C57BL/6 mouse model,q RT-PCR,immunofluorescence,and CFU results show that berberine can significantly reduce the content of H.pylori in mice.In addition,q RT-PCR and H&E staining results show that berberine can alleviate the inflammatory reaction in the gastric mucosa tissue of mice.3.Berberine effectively removes clinical drug-resistant strains of H.pylori in human gastric epithelial cells: In the H.pylori clinical drug-resistant isolate(532 and 536)infected HFE145 cell model,q RT-PCR results show that berberine can significantly reduce the content of H.pylori clinical drug-resistant strains in cells.II.Clarification that berberine removing H.pylori through targeting activation and repair of the autophagy-lysosome pathway1.Confirmation of the activation of the autophagy-lysosome pathway by berberine: By observing transmission electron microscopy,we found that compared with the control group,multiple bilayers and autophagosomes were observed in the H.pylori infection group.However,in berberine treatment group,multiple autophagolysosomes encapsulated and degraded bacterial bodies were observed.2.Confirmation that berberine removes H.pylori through the autophagy-lysosome pathway: By using autophagy inhibitors 3-MA,WM,and Baf-A1 to suppress key nodes in the autophagy pathway and si ATG5 and si BECN1 to knock down autophagy-related gene expression,we found that the content of H.pylori-specific 16 S r DNA in the berberine-treated group was significantly increased under H.pylori infection.3.Confirmation that berberine can restore the damaged autophagy-lysosome pathway during H.pylori infection: By Western blot analysis of related protein expression in cells and mice,we found that H.pylori infection resulted in a decrease in LC3B-II protein levels,an accumulation of p62 protein,and a stagnation of the conversion of CTSD protein from its precursor to its mature form.However,in the berberine-treated group,an increase in LC3B-II protein levels,a decrease in p62 protein levels,and a reversal of the stagnation of CTSD protein conversion to its mature form were observed.III.Exploring related molecules that can mediate the removal of H.pylori by berberine through the autophagy-lysosome pathway1.Validation in a cell model: We found that TLR4 and NLRP3 may be the molecules that mediate the activation and repair of the autophagy-lysosome pathway by berberine.We knocked down the expression of TLR4 and NLRP3 in cells by si RNA and detected the content of H.pylori by q RT-PCR.The results showed that the content of H.pylori in the TLR4 knockdown group did not change significantly compared to the normal cell group,while the content of H.pylori in the NLRP3 knockdown group increased significantly.In addition,Western blot was used to further detect the expression levels of relevant autophagy-lysosome proteins in cells.The results showed that there was no significant difference in the levels of LC3B-II,p62,and CTSD proteins in H.pylori-infected cells treated with or without berberine in the si NLRP3 transfection group.2.Validation in a gene knockout mouse model: First,we confirmed the successful construction of the gene knockout mouse model by genotyping the tail of the mouse.We used q RT-PCR,tissue immunofluorescence,and CFU detection to find that compared with the wild-type H.pylori-infected group,the content of H.pylori in the NLRP3 gene knockout mouse group did not change significantly regardless of whether berberine treatment was given.In addition,Western blot was used to detect the expression levels of relevant proteins in mouse gastric tissue.The results showed that in the NLRP3 gene knockout mouse,there was no significant difference in the levels of LC3B-II,p62,and CTSD proteins with or without berberine treatment.IV.Investigating and validating key downstream target genes activated and repaired by berberine1.Identification of differentially expressed autophagy-related genes(ARGs)by transcriptome sequencing: The four sets of data were integrated and analyzed by DESeq2 software to identify differentially expressed genes.Then,we screened for ARGs that were oppositely expressed in H.pylori-infected group and berberine-treated group.Finally,we selected 10 differentially expressed ARGs that were suppressed by H.pylori infection and restored by berberine treatment.We focused on these 10 potential downstream target genes for further screening and validation.2.Screening of key downstream target genes activated and repaired by berberine in autophagy-lysosome pathway: Based on multiple experiments at various transcriptional levels,we focused on EIF2AK4 as the key downstream target gene. Western blot analysis showed that the changes in protein expression of EIF2AK4 were consistent with changes in m RNA expression.3.Validation of key downstream target genes: In NLRP3 knockdown cells and mouse models,Western blot results showed that the function of berberine in regulating EIF2AK4 protein expression levels was inhibited.Then silencing of key downstream target gene EIF2AK4 expression using si RNA,q RT-PCR results showed that the intracellular removal of H.pylori by berberine after knockdown of EIF2AK4,Western blot results showed that the knockdown of EIF2AK4 inhibited the function of berberine in repairing the level of autophagy-associated lysosomal proteins.V.Elucidating the specific mechanism of berberine regulation of key downstream target genes1.Prediction of transcription factors that may activate key downstream target genes: We extracted and analyzed relevant data from NCBI,JASPAR,PROMO and STRING databases and predicted that STAT1 is a transcription factor that binds to the upstream promoter of EIF2AK4 through NLRP3-mediated nuclear translocation.2.Verification of specific regulatory mechanisms of transcription factors in upstream and downstream relationships: immunoprecipitation(Co-IP)results showed protein interactions between NLRP3 and STAT1.Immunofluorescence results show that berberine promotes STAT1 protein expression in the nucleus.Western blot results showed that NLRP3 could promote STAT1 protein entry into the nucleus.Chromatin immunoprecipitation(Ch IP)showed that in the berberine treated group STAT1 could directly bind to the promoter upstream of EIF2AK4.In addition,after further knockdown of STAT1 expression by si RNA,Western blot results showed that the upregulation of EIF2AK4 protein level by berberine was significantly inhibited in the knockdown STAT1 group.Conclusion:1.H.pylori can evade clearance by inhibiting the host cell autophagy-lysosome pathway and thus survive within the host cell.2.NLRP3 can mediate the removal of H.pylori by the autophagy-lysosome pathway induced by berberine.3.Berberine targets the NLRP3,promotes the translocation of transcription factor STAT1 into the nucleus,activates the downstream expression of EIF2AK4,repairs the autophagy-lysosome pathway,and further effectively removes intracellular H.pylori. |
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