| Background and Aim:As one of the most prevalent infectious pathogen in the stomach, Helicobacter pylori (Hp) is acknowledged to be the important environmental pathogenic factor of many disorders of the upper gastrointestinal tract, such as chronic gastritis, peptic ulcer disease, gastric cancer and MALT lymphoma. As the research moves along, the association of Hp infection and extra gastric manifestations attracts a lot of attention. For example the idiopathic thrombocytopenic purpura and iron deficiency anaemia have already been included in the consensus of Hp eradication. Previously, the mechanisms underlying Hp infection and extra gastric diseases probably include the production of a low-grade inflammatory state, induction of molecular mimicry and cross-immune reaction, the interference of nutrients or drugs absorption.There is growing epidemiological evidence for a potential association between Hp infection metabolic syndrome (MS). MS comprises a cluster of metabolic abnormalities including central obesity, impaired glucose tolerance, insulin resistance, dyslipidemia and hypertention, and it could strikingly increase the risk of diabetes, cardiovascular and cerebrovascular diseases. With the changes of lifestyle and diet structure (e.g. high fat and high calorie intake, sedentary lifestyle), the prevalence of MS is increasing worldwide which has become one of the serious public health issue for health threat. The imbalance of gut microbiota has currently demonstrated to play a vital role in triggering metabolic diseases such as obesity and MS. The infection of Hp which is a key member of the gastric microbiome is reported to alter the population structure of the gastric and intestinal microbiota. However, no study explores the association of Hp infection and gut microbiota with the development of MS so far. We therefore studied the relationship between Hp infection and MS given different dietary structure using Hp-infected animal models. Moreover, we observed the alteration of gut microbiota under different conditions and whether it plays a role in this process. The illustration of the issue will undoubtedly inspire new thoughts forMS prevention and also provide a theoretical basis for whether MS should be included in the Hp eradication indications.Materials and Methods:1. Hp-infected C57BL/6 mice models were built using two standard Hp strains namely SS1 and ATCC43504. The control group was given the sterile Brucella broth by gavage. Both Hp-infected groups and the control group were fed either a chow diet or high-fat diet (HFD).2. The growth and metabolism of the animals were monitored. The body weight, abdominal circumference and food and water intake in 24 hours were measured every two weeks. The energy intake and energy efficiency were then calculated.3. The glucose tolerance test and insulin tolerance test were performed both in the Hp-infected groups and control group after 12 and 24 weeks of different dietary intervention. Then the corresponding curve was drawn and the area under curve (AUC) was calculated.4. The blood biochemical indexes were tested both in the Hp-infected groups and control group after 12 and 24 weeks of different dietary intervention. The fast blood glucose was tested using glucose meter and the fasting serum insulin was tested by ELISA and then the HOMA-IR was calculated. The total cholesterol (TC) and triglyceride (TG) were tested using automatic biochemical analyzer. The serum hormones including leptin, resistin, glucose-dependent insulinotropic polypeptide (GIP), ghrelin and glucagon were tested by ELISA. The inflammatory cytokines including TNF-alpha, IL-lbeta and IL-6 were also tested using ELISA.5. Collect the fresh fecal samples of the Hp-infected groups and the control group after 12 and 24 weeks of different dietary intervention. Extract the bacterial DNA using the QIAamp DNA kit and then amplify the target 16S rRNA segment. After sample mixture, purification and database establishment it is ready for computer sequencing. Using the high-throughput sequencing platform Miseq from Illumina the V4 region of the samples were undergone pair-end sequencing. When the sequencing has finished, the bioinformatics methods were applied to analyze the structure of the gut microbiota.6. The animals were sacrificed after 12 and 24 weeks of different dietary intervention. The liver and visceral adipose tissue were isolated and weighed and then the mass index was calculated. The expression of the proteins of insulin signaling pathway IRS1/PI3K/Akt in the liver was determined by Western blot. The expression of the tight junction proteins in the ileal including ZO-1, occluding and claudinl was also detected by Western blot.Results:1. The effects of Hp infection on body weight and abdominal circumference after 12 and 24 weeks of different dietary intervention(1) The weight of the mice given HFD was significantly increased since week four compared with the control group (p<0.01). There was no obvious difference between Hp-infected group and the non-infected group.(2) The abdominal circumference of the mice given HFD was larger than the control group after 12 weeks of dietary intervention (p<0.01). In the HFD group, the abdominal circumference of the HpSS1-infected mice was larger than the non-infected subgroup (p<0.05), with no significant difference between the HpATCC43504-infected and non-infected mice (p>0.05). There was no obvious difference between Hp-infected group and the non-infected group when fed the chow diet (p>0.05).(3) The abdominal circumference of the mice given HFD continued to be larger than the control group after 24 weeks of dietary intervention (p<0.01). In the HFD group, the abdominal circumference of the HpSS1-infected mice was larger than the non-infected group (p<0.05), with no significant difference between the HpATCC43504-infected and non-infected mice (p>0.05). There was no obvious difference between Hp-infected group and the non-infected group when fed the chow diet (p>0.05).2. The effect of Hp infection on the weight and distribution of the fat tissue after 12 and 24 weeks of different dietary intervention(1) The content of visceral adipose tissue in the HFD mice was significantly higher than the control group with normal diet after 12 weeks of dietary intervention (p<0.01). In the HFD group, the content of visceral adipose tissue in the HpSS1-infected mice was higher than the non-infected subgroup especially the epididymal adipose tissue (p<0.01), with no significant difference between the HpATCC43504-infected and non-infected mice (p>0.05). There was no obvious difference between Hp-infected group and the non-infected group when fed the chow diet (p>0.05).(2) The visceral fat index of the HFD mice was higher than the control group given normal diet after 12 weeks of dietary intervention (p<0.01). In the HFD group, the visceral fat index was higher in the Hp-infected mice compared to the non-infected subgroup (p<0.01). There was no obvious difference between Hp-infected group and the non-infected group when fed the chow diet (p>0.05).(3) The content of epididymal adipose tissue in the HFD mice was significantly higher than the control group with normal diet after 24 weeks of dietary intervention (p<0.01). There was no obvious difference between Hp-infected group and non-infected group no matter given HFD or chow diet (p>0.05).(4) The epididymal fat index of the HFD mice was higher than the control group given normal diet after 24 weeks of dietary intervention (p<0.01). There was no obvious difference between Hp-infected group and non-infected group no matter given HFD or chow diet (p>0.05).3. The effect of Hp infection on energy metabolism after 12 and 24 weeks of different dietary intervention(1) The food intake of the HFD mice without Hp infection was decreased compared with the control group fed chow diet after 12 weeks (p<0.05), whereas the food intake of the Hp-infected mice given HFD were even lower (p<0.01). There was no obvious difference between Hp-infected and non-infected group given chow diet (p>0.05).(2) The water intake of the HFD mice without Hp infection was not different with the control group fed chow diet after 12 weeks (p>0.05), whereas the water intake of the Hp-infected mice was significantly decreased (p<0.01). There was no obvious difference between Hp-infected and non-infected group given chow diet (p>0.05).(3) The energy intake of the HFD mice without Hp infection was higher than the control group fed chow diet after 12 weeks (p<0.05), whereas the Hp-infected group fed HFD was not different from the control mice (p>0.05). The energy efficiency of both the HFD Hp-infected mice and non-infected mice were higher than the control group with normal diet (p<0.01). The energy intake and efficiency of the Hp-infected group were not different from the non-infected group (p>0.05).(4) The food intake of the HFD mice was not different from the control group with normal diet after 24 weeks (p>0.05), whereas the water intake was significantly decreased (p<0.05). The food intake of the mice with HpSS 1 infection and without infection given HFD was higher than the control group fed normal diet (p<0.05). The energy efficiency of HFD group without Hp infection rather than the Hp-infected mice was higher than the control group fed normal diet (p<0.01). There was no obvious difference between Hp-infected and non-infected group given chow diet (p>0.05).4. The effect of Hp infection on glucose and lipid metabolism after 12 and 24 weeks of different dietary intervention(1) The IPGTT showed that the AUC of the HFD mice was higher than the control group with normal diet after 12 weeks (p<0.01). In the HFD group, Hp-infected subgroup was not different from the non-infected mice. The IPITT showed that the AUC of the Hp-infected mice given HFD was higher than the non-infected mice (p<0.01). There was no obvious difference between Hp-infected and non-infected group given chow diet (p>0.05).(2) The fasting blood glucose of the HFD mice was higher than the control group with chow diet after 12 weeks (p<0.01). In the HFD group, the HpSS1-infected subgroup rather than the HpATCC43504-infected mice was higher than the non-infected mice (p<0.01). The fasting blood glucose was not different between Hp-infected and non-infected group given chow diet (p>0.05). The fasting insulin of the Hp-infected mice given HFD was higher than the control group with normal diet (p<0.05), while the HFD mice without Hp infection was not different from the control mice (p>0.05). The fasting insulin was not different between Hp-infected and non-infected group given chow diet (p>0.05). The HOMA-IR of the HFD mice without Hp infection was higher than the control group fed normal diet (p<0.05) while the Hp-infected HFD mice was even higher (p<0.01). In the HFD group, the HOMA-IR of the HpATCC43504-infected subgroup rather than those with HpSS1 infection was higher than the non-infected mice (p<0.01). The HOMA-IR was not different between Hp-infected and non-infected group given chow diet (p>0.05).(3) The IPGTT and IPITT showed that the AUC of the HFD mice was higher than the control group with normal diet after 24 weeks (p<0.01). There was no significant difference between Hp-infected and non-infected group no matter given HFD or normal diet (p>0.05). The fasting blood glucose of the HFD mice was higher than the control group with normal diet (p<0.01). In the HFD group, the fasting blood glucose of the HpSS1-infected subgroup was higher than the non-infected mice (p<0.01). The fasting blood glucose was not different between Hp-infected and non-infected group given chow diet (p>0.05). The fasting insulin of the HFD mice was higher than the control group fed chow diet (p<0.01), whereas there was no difference between Hp-infected and non-infected group (p>0.05). The HOMA-IR of the HFD mice was higher than the control group fed chow diet (p<0.01), whereas there was no difference between Hp-infected and non-infected group (p>0.05).(4) The serum TC of the HFD group was increased compared to the control group fed normal diet after 12 weeks (p<0.05), whereas there is no difference between Hp-infected and non-infected group (p>0.05). The serum TG of the HpSS1-infected mice was increased compared to the control group fed chow diet (p<0.01), whereas there was no difference between HFD group without Hp infection and the control group with chow diet (p>0.05). The serum TC of the HFD group was increased compared to the control group fed normal diet after 24 weeks (p<0.01), while the serum TG was not different between groups (p>0.05).5. The effect of Hp infection on inflammatory cytokines and hormones after 12 and 24 weeks of different dietary intervention(1) The serum level of TNF-α, IL-1β and IL-6 was not different between group after 12 weeks (p>0.05). The serum level of IL-6 was higher in the HpATCC43504-infected group compared to the control group fed chow diet after 24 weeks (p<0.05), while the serum level of TNF-α, IL-1β and IL-6 was not different between other groups (p>0.05).(2) The serum level of leptin of the HFD mice was higher than the control group fed chow diet after 12 weeks (p<0.01). In the chow diet group, the serum level of leptin of the HpATCC43504-infected rather than the HpSS1-infected mice was higher than the control group (p<0.05). The serum level of resistin of the HFD mice without Hp infection was not different from the control group fed chow diet, while the HFD Hp-infected mice was much higher (p<0.01). In the HFD group, the serum level of resistin of the Hp-infected subgroup was higher than the non-infected mice (p<0.01). The level of resistin was not different between Hp-infected and non-infected group when given chow diet (p>0.05). The level of GIP was higher in the Hp-infected HFD group rather than the non-infected group compared to the control group given chow diet (p<0.01). In the chow diet group, the level of GIP of the HpATCC43504-infected mice was higher than the control group (p<0.01). The level of ghrelin was higher in the HpATCC43504-infected HFD mice rather than the non-infected mice given HFD compared to the control group fed chow diet (p<0.05). In the HFD group, the level of ghrelin was higher in the Hp-infected subgroup than the non-infected group (p<0.05). The level of ghrelin was not different between Hp-infected and non-infected group when given normal diet (p>0.05). The level of glucagon was lower in the HpSS1-infected HFD mice rather than the non-infected group given HFD compared to the control group fed chow diet (p<0.05). The level of glucagon was not different between Hp-infected and non-infected group when given normal diet (p>0.05).(3) The level of leptin of the HFD mice was higher at 24 weeks than 12 weeks treatment (p<0.01), whereas there was no difference between Hp-infected and non-infected group (p>0.05). The level of resistin of the HFD mice without Hp infection was significantly increased at 24 weeks compared to 12 weeks and was not different from Hp-infected group given HFD (p>0.05), both of which were higher than the control group fed chow diet (p<0.05).6. The expression of the proteins of insulin signaling pathway IRS1/PI3K/Akt in the liver of the mice after 12 weeks of different dietary intervention(1) The expression of p-Akt (Ser473) in the Hp-infected group fed chow diet and the HFD group without Hp infection was not different from the control group fed chow diet (p>0.05). The expression of p-Akt (Ser473) in the Hp-infected HFD group was decreased compared to the HFD group without Hp infection and the control group fed chow diet (p<0.01).(2) The expression of p-IRS1 (Tyr896) in the Hp-infected group fed chow diet and the HFD group without Hp infection was not different from the control group fed chow diet (p>0.05). The expression of p-IRS1 (Tyr896) was lower in the Hp-infected HFD group compared to the control group fed chow diet (p<0.01). In the HFD group, the expression of p-IRS1 (Tyr896) was lower in the HpSS1-infected mice than the non-infected subgroup (p<0.01).7. The alteration of gut microbiota after 12 and 24 weeks of different dietary intervention and the expression of the tight junction proteins in the ileum(1) Hp infection had a greater impact on gut microbiota than the diet at 12 weeks. Both the PCoA analysis and the UPGMA methods based on Unifra revealed that the structure of gut microbiota was significantly different between Hp-infected group and the non-infected group given HFD (the contribution of PC1 was 40.51% and the contribution of PC3 was 9.33%). The a-diversity index was different between Hp-infected and non-infected group given HFD, with higher chao and shannon index with lower simpon index in the Hp-infected mice. At the phylum level, the proportion of sequences assigned to Firmicutes and Proteobacteria was significantly increased in the Hp-infected HFD group compared with non-infected group, whereas reads assigned to Bacteroidetes and Verrucomicrobia were reduced. At the family level, the relative abundance of Desulfovibrionaceae, Lachnospiraceae, Helicobacteraceae, Ruminococcaceae and Deferribacteraceae was higher in the Hp-infected HFD group, whereas the relative abundance of Alcaligenaceae, Veillonellaceae, Paraprevotellaceae and S24-7 was lower than the HFD group without Hp infection. At the genus level, the proportion of sequences assigned to Akkermansia and Sutterella was decreased while the reads assigned to Mucispirollum, Oscillospira and Helicobacter were increased compared to the HFD group without Hp infection. Furthermore, the LEfSe analysis revealed that there were different biomarker bacteria between these two groups. Besides, the relative abundance of Desulfovibrionaceae and Akkermansia was correlated with the HOMA-IR with negative correlation ofAkkermansia and positive correlation of Desulfovibrionaceae.(2) Fecal samples from Hp-infected mice fed chow diet formed a cluster that was different from those derived from control group at 12 week. The α-diversity index was not different between Hp-infected group and non-infected group given chow diet. At the phylum level, the relative abundance of Firmicutes was increased while the Bacteroidetes was decreased in the Hp-infected group given chow diet compared with the non-infected group. At the family level, the relative abundance of Deferribacteraceae, Helicobacteraceae and Spirochaetaceae was increased in the Hp-infected group than non-infected group given chow diet, while the relative abundance of Alcaligenaceae and Paraprevotellaceae was decreased. At the genus level, the proportion of Oscillospira, Mucispirillum and Treponema was increased in the Hp-infected group compared to non-infected group given chow diet, while the relative abundance of Sutterella and Parabacteroides was decreased.(3) The factor of diet had a greater impact on the composition of gut microbiota compared to Hp infection and took the dominant place after 24 weeks of different dietary intervention. The PCoA analysis showed that the fecal samples of the HFD mice clustered separately from the chow diet mice (the contribution of PC1 was 43.4% and the contribution of PC2 was 13.63%). The bray curtis clustering tree also achieved clustering between HFD group and chow diet group. At the phylum level, the relative abundance of Cyanobacteria and Verrucomicrobia was decreased in the HFD group without Hp infection compared with the control group fed chow diet. At the family level, the relative abundance of Bifidobacteriaceae, Erysipelotrichaceae, Lactobacillaceae and Verrucomicrobiaceae was decreased with increased abundance of Ruminococcaceae and Paraprevotellaceae in the HFD group without Hp infection compared to the control group fed chow diet. At the genus level, the relative abundance of Akkermansia, Bifidobacterium, Lactobacillus and Allobaculum was decreased with increased abundance of Oscillospira, Clostridium and AF12 in the HFD group without Hp infection compared to the control group fed normal diet.(4) The expression of intestinal tight junction protein ZO-1 was lower in the Hp-infected HFD group compared with other groups after 12 weeks of different dietary intervention. The expression of ZO-1 in the HFD group without Hp infection and Hp-infected group fed chow diet was not different from the control group fed chow diet. The expression of occludin was lower in the HpSS1-infected HFD group compared to the HFD group without Hp infection. The expression of occludin was lower in the HpATCC43504-infected HFD group compared to the control group fed chow diet although without difference with the non Hp-infected group given HFD. The HFD group without Hp infection also had a lower expression of occludin compared to the control group fed normal diet. The expression of occludin was not different between Hp-infected and non-infected group when given chow diet. The Hp-infected HFD group had a lower expression of claudinl compared to the HFD group without Hp infection. The expression of claudinl in the HFD group without Hp infection and Hp-infected group fed chow diet was not different from the control group fed chow diet.Conclusions:1. The effect of Hp infection on MS is associated with dietary structure and duration. Hp infection aggravates diet-induced insulin resistance, promotes fat distribution and accelerates the development of central obesity at the early stage of HFD. There is no effect of Hp infection on MS when given chow diet.2. The alteration of gut microbiota may play a role in the association of Hp infection with MS. The impact of Hp infection and diet on gut microbiota could manipulate and disturb the host metabolic homeostasis and finally lead to MS. |
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