Correlation Between Helicobacter Pylori Infection And Testosterone Along With Oxidative Stress Levels In Male Murines

Currently,couples suffering from infertility account for approximately 10%to 15%of the couples of childbearing ages in the world,and male infertility cases occupy about 50%.40%to 50%of the male cases are those of idiopathic infertility with unknown etiology.Research on the etiology of male infertility can provide a key theoretical basis for clinical decision-making of the disease.Helicobacter pylori(H.pylori)is a spiral,flagellated,and microaerobic gram-negative bacteria that infects more than half of the worlds population,and is closely associated with occurrence and development of the digestive system diseases such as chronic gastritis,peptic ulcer,MALT lymphoma and gastric cancer.Recent studies have suggested that H.pylori infection may cause a variety of parenteral diseases,but its relationship with male infertility remains undefined.ObjectiveThis study applied bioinformatics technology and animal models of H.pylori infection to explore the effect and mechanism of H.pylori infection on male reproductive function and provide a theoretical basis for the prevention and treatment of clinical male infertility.Methods1.Application of bioinformatics technology to analyze the relationship between H.pylori infection and reproductive function.①The miRNA microarray data sets GSE19769,GSE23877,GSE32174 and GSE54397 were downloaded from the GEO database.The four data sets contain a total of 31 H.pylori-negative tissue specimens and 41 H.pylori-positive tissue specimens;②The Limma package of R software was used to screen the differentially expressed miRNAs between H.pylori-positive and H.pylori-negative tissue specimens in each data set.The method of Benjamini and Hochberg was used to calculate the adjusted P value.The criterions for screening differentially expressed miRNAs were set as follows:adjusted P<0.05 and|log FC|>1.5;③The target genes of differentially expressed miRNA were searched in the miRTarBase database,and Cytoscape v3.8.2 was used to construct a miRNA-mRNA regulatory network;④String(www.string-db.org)was used to perform protein-protein interaction analysis on the target genes.Cytoscape v3.8.2 was applied to calculate the number of connections between each target gene node and the rest target gene nodes to determine the degree of contribution of the node in the entire network and draw a diagram;⑤R software was applied in gene ontology function enrichment analysis and drawing diagrams.2.Exploring the effect of H.pylori infection on the testis tissue of male mice through animal experiments.A C57/BL6 mouse model of acute H.pylori infection was constructed by gavage with this bacterium.After 2 weeks of H.pylori infection,testis and serum specimens were collected.The testicular tissue and serum specimens of Mongolian gerbil chronically infected with H.pyori(infected for 40 weeks)were collected from the College of Public Health,Zhengzhou University.Three oxidative stress indicators,namely malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were detected in the testes of the mice and gerbils.ELISA was used to determine serum testosterone levels and the Bax level as a testicular cell apoptosis indicator.Immunohistochemical technique was used to detect the expression of the inflammatory cytokine TNF-α in testis tissue.Results1.Bioinformatics analysis:①This study identified three differentially expressed miRNAs between the H.pylori-positive and H.pylori-negative tissue specimens,namely hsa-miR-200a,hsa-miR-200b and hsa-miR-196b.In the H.pylori-positive tissue specimens,the expression levels of hsa-miR-200a and hsa-miR-200b decreased,while the expression level of hsa-miR-196b increased;②The protein-protein interaction analysis of the target genes of the three differentially expressed miRNAs mentioned above identified STAT3 and MYC as the proteins encoded by the main target genes,which play important roles in physiological and pathological processes of the cell cycle and apoptosis regulation closely related to reproduction;③The GO function enrichment analysis showed that function of the target genes was enriched in the biological processes of response to oxidative stress,reproductive system and gland developments.The results suggest that H.pylori infection might affect the response to oxidative stress,tissue structure and function of the hosts’ testes.2.Animal experiment results:The urease tests of gastric tissue were positive in the H.pylori infected groups and negative in the control groups,indicating that the H.pylori infections were successful.Testicular tissue and serum specimens were collected for totaled 30 mice and 17 gerbils of the infected groups and the control groups.Results of the specimen inspection:①Testicular tissue oxidative stress indexes:The SOD expression level of H.pylori infected mice was lower than that of the control group(1105.77±194.43 vs 1260.29±197.93,P=0.047),the difference was of statistical significance.As for MDA[0.62(0.28-9.5)vs 1.46(0.34-3.38),P=0.227]and GSH-Px[126.44(107.32-200.28)vs 132.33(69.76-207.78),P=0.91]in the mice,no statistically significant difference was inferred between the infected and the control groups.There was no statistically significant difference in SOD[1052.83(677.76-1213.57)vs 986.91(546.74-1082.42),P=0.321],MDA(0.53±0.08 vs 0.55± 0.18,P=0.783)and GSH-Px(86.32 ±17.95 vs 73.72 ±18.48,P=0.176)between the H.pylori-infected gerbil group and the control group;②Detection of serum testosterone:The serum testosterone level of the H.pylori chronically infected gerbil group was lower than that of the control group(3.77 ± 0.95 vs 4.93 ± 0.97,P=0.026),and the difference was statistically significant.No statistically significant difference between the H.pylori acutely infected mice and the control group was detected(P=0.427);③The testicular cell apoptosis indicator was detected by ELISA:There was no significant difference in Bax between both the two mouse groups as well as the two gerbil groups(P>0.05);④HE staining of tissue sections:the morphology and structure of the testes in all groups were normal,and there was no significant difference between the gerbil groups as well as the mouse groups;⑤Immunohistochemical detection:No statistically significant difference in the expression levels of TNF-α in gerbil testis was detected between the infected and the control groups(0.044 ± 0.011 vs 0.039 ± 0.010,P=0.290).Conclusions1.The H.pylori-positive tissue specimens have three differentially expressed miRNAs compared to the H.pylori-negative tissue specimens,and the corresponding target gene function of the miRNAs is enriched in important biological processes related to reproduction such as the development of the reproductive system and response to oxidative stress.2.The levels of superoxide dismutase in the testis tissue of the C57/BL6 mouse models of acute H.pylori infection are lower than those of the control group,suggesting that acute H.pylori infection in humans may affect the level of oxidative stress in the testis tissue,thereby impairing male reproductive function.3.The serum testosterone levels of Mongolian gerbils chronically infected by H.pylori are statistically different from those of the uninfected control group,suggesting that chronic H.pylori infection in human may have impact on male reproductive function through the mechanism of down-regulating serum testosterone levels.4.H.pylori infection may involve the male reproductive system,cause changes in serum testosterone and testicular oxidative stress levels,and thereby affect male reproductive function,and thus control of H.pylori infection is expected to be a new thought on exploring the prevention and treatment strategy for male infertility.