The Role Of GPR1 In The Reproductive System Of LPS-Induced Depression Model In Mice

Depression is a common mental illness with high morbidity,mortality and disability.According to the World Health Organization,about 350 million people worldwide suffer from depression and last longer,adding to the burden of society and family.It is worth noting that women are twice as likely to suffer from depression as men,It suggests that there is a link between depression and female reproductive or reproductive diseases.Studies have shown that female reproductive system diseases（such as endometrial cancer,polycystic ovary syndrome,etc.）are often accompanied by an increase in the prevalence of depression,although the cause of this increased prevalence is unclear,but obesity,insulin resistance,Estrogen elevation,infertility,HPA axis abnormalities and inflammation may play a role.However,it is unclear whether depression will cause changes in reproductive system function.In recent years,the causal relationship between depression and female reproductive system-related diseases has been controversial.According to related literature reports,GPR1 mRNA is highly expressed in the ventricles of the hypothalamus and reproductive system of the central nervous system.This study used lipopolysaccharide（LPS）intraperitoneal injection to establish a female mouse depression model,and detected changes in reproductive related indicators in mice with depression,and try to use GPR1 antagonist peptide G5 to explore the effect of GPR1 on depression-like indicators and reproductive hormone secretion in mice with depression.The content and results of this study are as follows:（1）Depression is related to the secretion activity of the hypothalamus.We take the hypothalamus of normal mice for paraffin embedding and sectioning,and immunohistochemically stain the hypothalamus of normal female mice to explore whether there is GPR1 expression in the hypothalamus.The results showed that GPR1 was significantly expressed in the cytoplasm of normal hypothalamic cells in normal female mice,suggesting that GPR1 may be associated with certain functions of the hypothalamus.（2）To further investigate whether GPR1 is associated with HPA and HPO axis activity,we used paraffin section immunofluorescence to co-stain GPR1 and CRF in the hypothalamus of normal mice.The results showed that GPR1 and CRF were co-expressed in the cytoplasm of most cells in the posterior hypothalamus of normal female mice.we found that GPR1 and GnRH are also co-expressed in the cytoplasm of most cells in the anterior hypothalamus of normal female mice.This indicates that GPR1 may be involved in the secretory function of hypothalamic CRF and GnRH.（3）In order to explore whether GPR1 plays a role in depression,we used lipopolysaccharide to establish a mouse model of depression and used the laboratory-selected GPR1-specific antagonist peptide G5,while using the classic antidepressant fluoxetine as a positive control drug.By calculation,three concentrations of low（2.2585mg/kg）,medium（22.585mg/kg）and high（225.85mg/kg）G5 were set,wherein the medium concentration of G5 and fluoxetine were consistent in the same number of effective molecules per unit volume.We found that G5-treated depressive mice showed weight recovery,increased sucrose preference,and reduced duration of inactivity in swimming,suggesting that G5 may play a role in relieving depression by inhibiting GPR1,and from behavioral testing As a result,medium-concentration G5 is more effective than Flu.（4）In order to further verify the success of the depression model at the molecular level,it is also explored how the GPR1 antagonist peptide G5 exerts antidepressant effects and whether depression leads to changes in reproductive system function.We analyzed the tissue after the end of the sucrose test and analyzed it from three levels:inflammation,nerve and reproduction.The inflammatory factors in the serum and the changes of steroid hormones in the ovary were detected by radioimmunoassay.The changes of neurotransmitters in hippocampus and prefrontal cortex were detected by high performance liquid chromatography-tandem mass spectrometry.The changes of brain-derived neurotrophic factor（BDNF）levels were detected by enzyme-linked immunosorbent assay（ELISA）,and the changes of serum corticosterone levels after 4 hours of LPS injection were measured by enzyme-linked immunosorbent assay.The results showed that intraperitoneal injection of lipopolysaccharide in mice caused increased levels of Tumor Necrosis Factor-α（TNF-α）,interleukin-6（IL-6）,and interleukin-1β（IL-1β）,decreased levels of neurotransmitters Gamma-Amino Butyric acid（GABA）and 5-HT（5-hydroxytryptamine）in the hippocampus and prefrontal cortex,and decreased levels of BDNF in the hippocampus.G5 alleviated the changes in molecular levels caused by depression,suggesting that G5 may exert antidepressant effects through GPR1.Further detection of changes in serum corticosterone after 4 hours of LPS injection showed that the level of CORT in the depression model group was significantly increased,and the low and medium concentrations of G5 decreased the level of CORT,which once again proved that G5 may have the effect of relieving depression.More importantly,compared with the control group,the levels of P₄ and E₂ in the serum of the depression model mice were decreased.Correspondingly,the level of the enzyme cyp21a1 mRNA involved in progesterone metabolism was significantly increased,and the level of Aromatase mRNA involved in the synthesis of estrogen is also reduced accordingly.This indicates that LPS-induced depression affects the synthesis of P₄ and E₂in mice and the expression of related steroid synthase,while G5 can alleviate the changes in reproductive hormones and related enzyme genes caused by depression.In summary,this paper demonstrates for the first time that GPR1 is co-expressed with GnRH and CRF in the hypothalamus,indicating a role of GPR1 in the bridge between depression and reproduction.In addition,we also applied GPR1 antagonist peptide G5 for the first time to explore the effect of GPR1 on LPS-induced depression in mice.Experiments have shown that moderate G5（22.5mg/kg）has a certain effect on antidepressant treatment and improving reproductive hormone changes caused by depression.This suggests that there may be a link between depression and reproductive system diseases through a common target GPR1.