Study On The Role Of IL-17 In The Inflammation Of Mouse Adipose Tissue Induced By Bisphenol A

Background As an important raw material for chemical industry,bisphenol A?BPA?is produced in large quantities and used widely around the world.Generally,BPA is used as an additive in many kinds of plastics.Hence we can find it in canned food,feeding bottle,water bottle,seal gum,glasses and many other daily necessities.The wide use of BPA causes many pollution problems for its easily release to the food and water when it is heated or in contact with acid or oil.Numerous studies have showed that people were exposed to BPA extensively.BPA is known as a typical kind of environmental endocrine-disrupting chemicals.Epidemiological studies and animal experiments showed a close correlation link between BPA and obesity.Obesity can cause a chronic low-grade inflammatory state which is related to the inflammation in adipose tissue.And inflammation in adipose tissue was considered to be the key driving factor of obesity related diseases.The accumulation and activation of immune cells were closely related to the inflammation in adipose tissue.IL-17,which mainly secreted by Th17 cell and plays a key role in the development of inflammation and immune-related diseases,has been a hot topic for its close relationship with inflammation.However the role of IL-17 in the inflammation of mouse adipose tissue induced by BPA is still not clear.Objective To evaluate the effects of BPA on the inflammation,IL-17 expression and frequency of Th17 cells in adipose tissue through animal experiment.Then to assess the effects of IL-17 antibody on the inflammation in mouse adipose tissue induced by BPA.And provide experimental data for the development of prevention measures and strategies for health hazards of BPA exposure.Method The first part: study on expression of IL-17 in adipose tissue inflammation induced by BPA exposure 4 week-old C57BL/6J wild male mice were divided into two groups: normal diet group?ND?and high fat diet?HFD?group.Then each group was subdivided into vehicle control group,10nM BPA group,100nM BPA group and 1000nM BPA group.There were 24 mice in every group and 6 mice were sacrificed after exposing BPA for 8,12,16 and 20 weeks,respectively.The epididymal fat pad was obtained for experiment.The second part: The effects of IL-17 antibody on inflammation in adipose tissue.4 week-old C57BL/6J wild male mice were fed on HFD and were randomly divided into BPA unexposed group and BPA exposed group.The BPA dose was 1000nM.Then each group was subdivided into vehicle control group,IgG isotype control group and IL-17 antibody treated group.There were 8 mice in every group.Mice in IgG isotype control group and IL-17 antibody treated group were administered 100?g IgG and 100?g IL-17 antibody respectively by intraperitoneal injection weekly.Mice were sacrificed after exposing for 8 weeks and the epididymal fat pad was obtained for experiment.Histopathology was used to assess the accumulation of inflammatory cells.Immunological histological chemistry?IHC?and enzyme linked immunosorbent assay?ELISA?were used to assess the inflammatory cytokines expression of IL-17,TNF-?,IL-1? and IL-6in adipose tissue.Real time-polymerase chain reaction?RT-PCR?were used to detect the IL-17 m RNA expression in adipose tissue.FCM was used to evaluate the frequency of Th17 cells in adipose tissue.Results 1.Effects of BPA exposure on mouse body weight.Compared with vehicle control group,BPA exposure increased mouse body weight in a time and dose-depend way.In ND group,compared with vehicle control group,the increasing of mouse body weight began to have statistical significance after 12 weeks in 1000nM BPA group and after 19 weeks in 100nM BPA group?P<0.05?.In HFD group,compared with vehicle control group,the increasing of mouse body weight began to have statistical significance after 11 weeks in 1000nM BPA group and after 15 weeks in 100nM BPA group?P<0.05?.Compared with ND group,the effect of BPA in HFD group was stronger.2.Effects of BPA exposure on inflammation in adipose tissue.2.1.Histopathology results of adipose tissue.Compared with ND group,inflammatory cell infiltration was more severe in HFD group.Compared with vehicle control group,BPA exposure enhanced inflammatory cell infiltration in a time and dose-depend way.2.2.IHC results of TNF-?,IL-1? and IL-6 expression in adipose tissue.Compared with ND group,TNF-?,IL-1? and IL-6expression were higher in HFD group.Compared with vehicle control group,BPA exposure increased the expression of TNF-?,IL-1? and IL-6 in a time and dose-depend way.2.3.ELISA results of TNF-?,IL-1? and IL-6 expression in adipose tissue.Compared with ND group,TNF-?,IL-1? and IL-6 expression were higher in HFD group.Compared with vehicle control group,BPA exposure increased the expression of TNF-?,IL-1? and IL-6 in a time and dose-depend way.In ND group,compared with vehicle control group,the increasing of TNF-? and IL-6 expression began to have statistical significance after 12 weeks in 1000nM BPA group and 100nM BPA group and after 16 weeks in 10nM BPA group?P<0.05?.The increasing of IL-1?expression began to have statistical significance after 12 weeks in 1000nM BPA group,after 16 weeks in 100nM BPA group and after 20 weeks in 10nM BPA group?P<0.05?.In HFD group,compared with vehicle control group,the increasing of TNF-? expression began to have statistical significance after 8 weeks in 1000nM BPA group and after 12 weeks in 100nM BPA group and 10nM BPA group?P<0.05?.The increasing of IL-6 expression began to have statistical significance after 8 weeks in 1000nM BPA group and 100nM BPA group and after 12 weeks in 10nM BPA group?P<0.05?.The increasing of IL-1? expression began to have statistical significance after 8 weeks in 1000nM BPA group,after 12 weeks100nM BPA group and after 16 weeks in 10nM BPA group?P<0.05?.3.Effects of BPA exposure on IL-17 expression in adipose tissue.Compared with the ND group,HFD increased the expression of IL-17.Compared with vehicle control group,BPA exposure increased IL-17 expression in a time and dose-depend way.According to the result of RT-PCR and compared with the ND group,HFD increased the expression of IL-17 m RNA.Compared with vehicle control group,BPA exposure increased IL-17 m RNA expression in a time and dose-depend way.In ND group,compared with vehicle control group,the increasing of IL-17 m RNA expression began to have statistical significance after 12 weeks in 1000nM BPA group and 100nM BPA group and after 20 weeks in 10nM BPA group?P<0.05?.In HFD group,compared with vehicle control group,the increasing of IL-17 m RNA began to have statistical significance after 12 weeks in 1000nM BPA group and 100nM BPA group and after 16 weeks in 10nM BPA group?P<0.05?.Compared with the ND group,ELISA result showed HFD increased IL-17 expression.Compared with vehicle control group,BPA exposure increased IL-17 expression in a time and dose-depend way.In ND group,compared with vehicle control group,the increasing of IL-17 m RNA expression began to have statistical significance after 12 weeks in 1000nM BPA group and 100nM BPA group and after 20 weeks in 10nM BPA group?P<0.05?.In HFD group,compared with vehicle control group,the increasing of IL-17 m RNA began to have statistical significance after 12 weeks in 1000nM and 100nM BPA group and after 16 weeks in 10nM BPA group?P<0.05?.4.Effects of BPA exposure on Th17 cell frequency in adipose tissue.Compared with ND group,HFD increased Th17 cell frequency.Compared with vehicle control group,BPA exposuree increased Th17 cell frequency in a time and dose-depend way.In ND group,compared with vehicle control group,the increasing of Th17 cell frequency began to have statistical significance after 12 weeks in 1000nM BPA group,after 16 weeks in 100nM BPA group and after 20 weeks in 10nM BPA group?P<0.05?.In HFD group,compared with vehicle control group,the increasing of Th17 cell frequency began to have statistical significance after 12 weeks in 1000nM BPA group and 100nM BPA group and after 20 weeks in 10nM BPA group?P<0.05?.5.Effects of IL-17 antibody on IL-17 expression in adipose tissue.IHC results showed that BPA exposure increased the expression of IL-17.Compared with their own vehicle control group,IgG administration showed no significant effect on IL-17 expression.Therefore,compared with their own IgG control group,IL-17 antibody significantly decreased the expression of IL-17.6.Effects of IL-17 antibody on Th17 cell frequency in adipose tissue.FCM results showed that BPA exposure increased Th17 cell frequency.Compared with their own vehicle control group,IgG administration showed no significant effect on Th17 cell frequency.Compared with their own IgG control group,although IL-17 antibody decreased Th17 cell frequency,there was no statistical significance?P>0.05?.Compared with HFD group,BPA exposure increased Th17 cell frequency and there was statistical significance at 16^th week?P<0.05?.7.IL-17 antibody inhibited the increasing of mouse body weight and epididymal fat pad coefficient induced by BPA.Compared with their own vehicle control group,IgG administration showed no significant effect on mouse body weight and epididymal fat pad coefficient.Compared with their own IgG control group,IL-17 antibody decreased mouse body weight,there was statistical significance in 1000nM BPA exposed group at 9^th,10^th,11^th and 16^th week?P<0.05?.Compared with HFD group,BPA exposure increased mouse body weight,there was statistical significance at 15^th and 16^th week?P<0.05?.IL-17 antibody decreased mouse epididymal fat pad coefficient.There was no statistical significance?P>0.05?in BPA unexposed group and there was statistical significance?P<0.05?in BPA exposed group.Compared with HFD group,BPA exposure increased mouse epididymal fat pad coefficient,there was statistical significance at 16^th week?P<0.05?.8.IL-17 antibody alleviated inflammation in adipose tissue induced by BPA Compared with their own vehicle control group,IgG administration showed no significant effect on inflammatory cell infiltration in adipose tissue.Compared with their own IgG control group,IL-17 antibody alleviated inflammatory cell infiltration in adipose tissue.IHC results showed BPA increased the expression of TNF-?,IL-1? and IL-6.Compared with their own vehicle control group,IgG administration showed no significant effect on the expression of TNF-?,IL-1? and IL-6.Compared with their own IgG control group,IL-17 antibody decreased the expression of TNF-?,IL-1? and IL-6.Conclusion 1.BPA exposure promoted inflammatory cell infiltration and the expression of pro-inflammatory cytokines in adipose tissue.2.BPA exposure increased IL-17 expression and Th17 cell frequency in adipose tissue.3.IL-17 antibody inhibited the increasing of IL-17 expression,but showed limited effect on Th17 cell frequency in adipose tissue induced by BPA.4.IL-17 antibody inhibited inflammatory cell infiltration and the expression of pro-inflammatory cytokines in adipose tissue induced by BPA.