| Background and ObjectiveObesity and allergic diseases are two types of chronic non-communicable diseases which are difficult to cure,recurrent and lead to serious socio-economic and health burden.The World Health Organization has also indicated that obesity and allergic diseases will be the most concerned public health problems in this century.With the improvement of quality of life,the prevalence of obesity and allergic diseases in China has also increased rapidly.The cohort study has pointed out that due to the differences in diet and genetic background,the Chinese mainly developed into central obesity with the characteristics of less weight gain and severe visceral fat accumulation compared with Obese patients in western countries.Epidemiological studies have indicated that obesity and allergic diseases were risk factors for each other’s comorbidity,however,the interaction and mechanism of the two diseases have not been clarified.Therefore,further analysis of the relationship and pathogenesis of the comorbidity may be a new method to prevent the development of obesity and allergic diseases.Gut microbiota has been proved to play an important role in the regulation of obesity and allergic diseases,however,there is still a lack of research on gut microbiota in the comorbidity of obesity and allergic diseases.This study used animal experiments to establish an animal model of central obesity and to identify the metabolic function and gut microbiota characteristics of central obesity mice.On this basis,to clarify the correlation between central obesity and allergic disease and the potential function and mechanism of gut microbiota and their metabolites in the development of the comorbidity,this study used post-central obesity allergic mice and post-allergy central obese mice to identify the changes of metabolism,hormone secretion,allergic reaction,immune response and histopathology and to indicate the key factors and correlation in the dynamic development of the comorbidity.Meanwhile study also determined the dynamic changes in gut microbiota,metabolites of these microbiota（short-chain fatty acids and bile acids）and the expression of the related metabolites receptors to clarify the potential function and mechanism of gut microbiota and their metabolites in the development of the comorbidity which may contribute to provide reference for the early prevention of obesity and allergic disease comorbidity.MethodsThis study was consisted of three parts.High-fat diet was used to induce central obesity BALB/c mice and the characteristics of the central obesity mouse model and the detailed changes of gut microbiota were investigated in the first part;in the second part OVA and AL（OH）3 was used to induce allergic mice to observe the gender differences of inflammation and allergy in allergic mice;then HFD and OVA+AL（OH）3were used to induce the post-central obesity allergic mice to indicate the effects of obesity on allergic reaction,immune response,gut microbiota,gut microbiota metabolites and expression of the related receptors and to clarify the effects of central obesity on the development of allergic diseases and the regulatory role of gut microbiota in the dynamic development of comorbidity;in the third part,OVA+AL（OH）3 and HFD were used to induce post-allergy central obese mice to indicate the effects of allergy on metabolism,leptin and insulin secretion,histopathology,gut microbiota,gut microbiota metabolites and expression of the related receptors in central obese mice and to clarify the effects of allergy on the development of central obesity and the regulatory role of gut microbiota in the dynamic development of comorbidity.The detailed experimental design in each part were listed as follows:（1）Three-week-old specific pathogen-free BALB/c mice were randomly grouped into normal diet male group,normal diet female group,high-fat diet male group and high-fat diet female group.After twelve-week normal diet or high-fat diet,fasting blood glucose and oral glucose tolerance of BALB/c mice were measured then these mice were sacrificed to collect serum,fecal,liver,spleen and visceral adipose tissue.ELISA was used to detect the serum leptin and insulin.Insulin resistance index was calculated from FBG and serum insulin.Automatic biochemical analyzer was used to detect serum level of total cholesterol,triglyceride,high-density lipoprotein cholesterol,low-density lipoprotein cholesterol.Hematoxylin-Eosin and Oil red O were used to stain the adipose tissue and liver tissue respectively.16S r RNA sequencing was used to detect the diversity,structure and functional prediction of fecal intestinal microbiota.（2）First,specific pathogen-free BALB/c mice were randomly grouped into Control male group,Control female group,OVA male group and OVA female group.The body weight of mice was measured before and after OVA sensitization and the serum Ig E and OVA specific Ig E of allergic mice were measured by ELISA.Then RT-q PCR was used to detect the spleen and visceral adipose tissue TNF-α、IFN-γ、IL-6、IL-10 and IL-17 m RNA expression.In the comorbidity study,Specific pathogen-free BALB/c mice were randomly grouped into Control group,OVA group and high-fat diet+OVA group（HOA group）.Mice in Control group and OVA group were fed with normal diet and those in HOA group were fed with high-fat diet.After 12 weeks of high-fat diet,mice in OVA group and HOA group were sensitized with OVA at the first,third,fourth and fifth week.Then mice were sacrificed to collect serum,fecal,liver,spleen and visceral adipose tissue.ELISA was used to detect the serum level of Ig E and OVA-Ig E;RT-q PCR was used to detect the spleen and visceral adipose tissue TNF-α、IFN-γ、IL-6、IL-10 and IL-17 m RNA expression;flow cytometry was used to detect the proportion of CD4+CD25+FOXP3+Treg in spleen;16S r RNA sequencing was used to detect mice gut microbiota;GC and UPLC-MS were used to detect the SCFAs and BAs in mice fecal;western blot was used to detect the expression of GPR41,GPR43,GPR109A,FXR and TGR5 in mice spleen.（3）Specific pathogen-free BALB/c mice were randomly grouped into Control group,high-fat diet group and OVA+high-fat diet group（HOA group）.Mice in HOA group were sensitized with OVA at the first,third,fourth and fifth week.At the sixth week,blood was sampled from the mice tail and the serum level of Ig E and OVA-Ig E were measured.Then 5 mice in each group were sacrificed to detect TNF-α、IL-6 and IL-10 m RNA expression in colon.Meanwhile at the sixth week,mice in Control group were fed with normal diet and others were fed with high-fat diet.After 12 weeks of high-fat diet or normal diet,fasting blood glucose and oral glucose tolerance were measured then mice were sacrificed to collect serum,fecal,liver,spleen and visceral adipose tissue.Mice spleen index,liver index and visceral adipose weight were measured.Automatic biochemical analyzer was used to detect serum level of total cholesterol,triglyceride,high-density lipoprotein cholesterol,low-density lipoprotein cholesterol;Enzvmati C assay was used to detect liver level of TG and TC;ELISA was used to detect the serum leptin and insulin;insulin resistance index was calculated from FBG and serum insulin;hematoxylin-Eosin was used to stain the adipose tissue and to measure the area of adipocyte;16S r RNA sequencing was used to detect the diversity and structure;GC and UPLC-MS were used to detect the SCFAs and BAs in mice fecal;western blot was used to detect the expression of GPR41,GPR43,GPR109A,FXR and TGR5 in mice liver.Results（1）Male mice fed with high-fat diet were observed with significant higher body weight,visceral adipose weight,FBG,OGTT,AUC,TC,leptin,insulin,insulin resistance index,fat accumulation in liver and adipose tissue and with lower serum level of TG.However there were no difference in body weight,visceral adipose weight,FBG,OGTT,AUC,leptin,insulin and insulin resistance index in female mice fed with high-fat diet.Compared with female mice,fat accumulation in adipose tissue and liver tissue was more significant in male mice fed with high-fat diet.Although the high-fat diet did not affect gut microbitoa diversity significantly,the high-fat diet induced more drastic changes in gut microbiota composition and microbial taxa in male mice.Meanwhile the functional prediction of gut microbiota based on Tax4fun also indicated that male mice fed with high fat-diet showed more significant changes in expression of immune system,energy metabolism,carbohydrate metabolism,lipid metabolism,and endocrine system.（2）There were no significant difference in body weight of allergic mice,however,the levels of serum Ig E and OVA specific Ig E were increased and the relative expression of cytokines in spleen changed significantly.Meanwhile there was no gender difference in the above characteristics.In the study of the comorbidity,compared with mice in control group,the expression of IL-6 and IL-17 in adipose tissue and spleen tissue,the expression of TNF-αin adipose tissue and the expression of IL-10 in spleen tissue were significant increased in allergic mice.However the proportion of Treg cells in allergic mice spleen did not change.Compared with mice in control group,post-central obesity allergic mice showed significant increase in expression of TNF-α、IFN-γand IL-17 in both adipose tissue and spleen tissue,however,there was no difference in expression of IL-6 in liver tissue and IL-10 in adipose tissue.Meanwhile compared with allergic mice,post-central obesity allergic mice showed higher level of serum total Ig E and lower level of serum OVA-Ig E.Additionally,the proportion of Treg cells in post-central obesity allergic mice also showed significant increase.There were significant differences in the composition of gut microbiota among the Control group,OVA group and HOA group.The gut microbiotaα-diversity increased and the fecal SCFAs level and the expression of GPR41 and GPR43 receptors decreased in allergic mice,however,there was no significant change in bile acids metabolism and expression of GPR109A,TGR5and FXR receptor in allergic mice.Post-central obesity allergic mice were observed further decrease in fecal SCFAs level and in the expression of GPR41 and GPR43.Meanwhile mice in HOA group were also observed significant increase in fecal LCA、7-keto LCA、12-keto LCA、DCA、UDCA、HDCA、α-MCA、UCA、β-MCA、CA、GDCA、TDCA and T-α-MCA.However the expression of FXR and TGR5 did not change in the post-central obesity allergic mice liver.（3）There was no significant change in the secretion of cytokines in the colon tissue of mice after OVA stimulation.However there was significant ascites in the abdominal cavity of allergic mice in the central obesity group after 12 weeks of high-fat diet.Allergy resulted in increase of liver index and spleen index but decrease of body weight and visceral adipose weight in central obese mice.Compared with central obese mice,allergy significant decreased obese mice TG,TC,HDL-c,liver TG and insulin resistance index with no influence on OGTT,AUC,area of adipocyte and serum level of leptin and insulin.There was no difference in gut microbiota diversity and composition,content of fecal propionic acid and butyric acid and expression of GPR41 and GPR43 in liver between HFD group and OHF group.Importantly,allergy resulted in increase of acetic acid and expression of GPR109A in central obese mice.In addition,post-allergy central obese mice also observed significant increase of fecal DHCA,6,7-diketo LCA,GDCA,TCA,T-alpha-MCA,T-β-MCA and FXR expression.Conclusion（1）Centrally obese mice gained less weight and had severe visceral fat accumulation and disturbances in glycolipid metabolism and hormone secretion.Central obesity in BALB/c mice induced by high-fat diet has significant gender difference,and this gender sensitivity may be due to the differences in composition,structure and functional expression of gut microbiota and the stability of those in BALB/c female and male mice fed with high-fat diet.Thus gut microbiota plays an important role in the development of central obesity and may contribute to the regulation of metabolic function and energy balance.（2）The body weight,serum level of Ig E and OVA-specific Ig E and spleen cytokines of allergic mice induced by OVA have no gender difference.In the study of comorbidity,the dysbiosis of gut microbiota,the decrease of short chain fatty acids and expression of GPRs receptor caused by central obesity can aggravate the allergy in mice by affecting the level of cytokines and damaging the function of Treg cells and promote the development of allergic diseases.Thus in the allergic diseases promoted by obesity,gut microbiota and their metabolites may be the key factors to regulate the development of these disease.（3）Early allergy led to the decrease of body weight and visceral fat and severe hydroperitoneum,however,early allergy did not aggravate the disorder of glucose and lipid metabolism in central obese mice.Allergy did not affect the diversity,composition and structure of gut microbiota in post-allergy central obese mice while allergy could change the metabolism of acetic acid,BAs and vitamin B3 and promote the expression of GPR109A and FXR in central obese mice liver via independence of gut microbiota composition.Therefore,allergic diseases may regulate the development of obesity mainly through metabolites of gut microbiota rather than the composition and structure of gut microbiota and gut microbiota metabolites may be the key factor of allergy-mediated obesity comorbidities.In this study,animal experiments were used to construct central obesity mice model,post-central obesity allergic mice model and post-allergy central obese mice model.These animal models were used to indicate the effects of central obesity on allergic reaction,immune response,gut microbiota,gut microbiota metabolites and expression of the related receptors and the effects of allergy on metabolism function,leptin and insulin secretion,histopathology,gut microbiota,gut microbiota metabolites and expression of the related receptors.The results have proved that central obesity may impair the allergy and immune function and to aggravate and promote the development of allergic diseases mainly via altering the gut microbiota and level of SCFAs.Meanwhile the study also indicated that allergy may partly alter the gut microbiota metabolites and the expression of related receptors via independence of gut microbiota composition to regulate the function of metabolism.These results suggested that gut microbiota and their metabolites may be the key roles for regulating the development of allergy-mediated obesity diseases and obesity-mediated allergic diseases and may provide new ideas for the prevention and treatment of these two kinds of chronic non-communicable diseases. |
