Effects Of Systemic Inflammatory Response Induced By Lipopolysaccharide On Lactation In Mice

Mammals have reduced immunity after childbirth,and are prone to infection with bacteria such as Escherichia coli,which can cause diseases such as mastitis,endometritis,and vaginitis,causing systemic inflammatory response,leading to decreased milk production.The mechanism of mastitis inhibiting lactation has been deeply studied,but there are few studies on the effect of other inflammation on lactation.Therefore,in this experiment,lipopolysaccharide(LPS),the main virulence factor of gram-negative bacteria,is injected intraperitoneally to induce systemic inflammatory response in mice at the early stage of lactation.We combined with RNA-seq to explore the way that inflammation affects lactation from the point of view of hormones,the balance of oxidation and antioxidation and breast tissue structure.Systemic inflammatory response was induced in mice on the 7th day of lactation by intraperitoneal injection of LPS at doses of 2.5,5,and 10mg/kg body weight,with mice injected with equal volume physiological saline as the control.Six hours after injection,the serum of mice was collected to detect the levels of Tumornecrosisfactor-α(TNF-α),Interleukin-1β(IL-1β)and Interleukin-6(IL-6).The mammary gland in area 4 was collected to make pathological sections.The visceral organs such as heart,liver,spleen,lung and kidney were weighed and the organ index was calculated.The body weight,feed intake and litter weight of mice on day 4-17 of lactation were recorded,and the amount of lactation per hour and total lactation were calculated.Select the appropriate induction dose according to the degree of inflammation and the effect on the mammary.The results showed that 5 mg/kg body weight LPS can induce obvious systemic inflammatory symptoms in mice,increase spleen index and lung index,increase serum proinflammatory cytokines and decrease milk production,so 5 mg/kg body weight LPS was used as the appropriate dose for further study.In the process of LPS dose selection,it was found that the food intake of mice decreased sharply,and there was a close relationship between milk yield and feed intake,so the feeding intervention group was set up to study the direct effect of inflammation more accurately.The body weight,feed intake and litter weight of maternal rats on day 4-17 of lactation were recorded,and the effects of milk yield per hour and total lactation on lactation performance were calculated.Serum samples were collected to detect the levels of inflammation-related factors such as TNF-α,IL-1β,IL-6 and hs-CRP,lactation-related hormones such as prolactin(PRL)and growth hormone(GH),Oxidative antioxidant indicators such as superoxide dismutase(SOD)activity and malondialdehyde(MDA)content.Mammary tissue was collected and paraffin section technique was used to observe breast structure.Immunofluorescence technique was used to observe the localization of breast tight junction protein Claudin-1 and ZO-1.Westernblot technique was used to analyze the expression of tight junction protein.Transcriptome sequencing was used to screen differential genes and enrichment pathways,real-time fluorescence quantitative PCR was used to verify the gene level of differential genes,and Westernblot technology was used to verify the protein level of signal pathways of significant enrichment of differential genes.The results showed that:(1)Lactation performance: compared with CG,the single hour milk yield and total milk yield of IG and RG mice decreased significantly,and the total milk yield of IG mice decreased significantly compared with RG.(2)Serum detection results:compared with CG,the levels of pro-inflammatory cytokines such as TNF-α,IL-1β and IL-6in serum of IG and RG mice increased significantly,the content of PRL in IG 6 h,IG 24 h and RG 24 h increased significantly,the content of GH decreased,PRL content increased in RG 6 h,SOD activity decreased and MDA content increased in IG 6 h,IG 24 h and RG 24 h,SOD activity decreased in RG 6 h.Compared with RG,the levels of TNF-α and hs-CRP at 6h in IG increased significantly,the levels of TNF-α,IL-1β,IL-6 and hs-CRP at 24 h in IG increased significantly,the levels of GH at 6 h in IG and 24 h in IG decreased significantly,the level of PRL in IG 24 h increased significantly,SOD activity decreased significantly in IG 6 h and IG 24 h,and MDA content increased significantly.(3)Tissue structure of mammary gland: compared with CG,a large number of mammary epithelial cells were exfoliated and inflammatory cells infiltrated in alveolar cavityin IG 6 h and IG 24 h,and acinar filling area was reduced in RG 6 h and RG 24 h.Immunofluorescence results showed that Claudin-1 and ZO-1 proteins were uniformly distributed around mammary ducts in CG and RG mice,RG was densely distributed and IG distribution was chaotic.The results of Western blot showed that compared with CG,the expression of Claudin-1 protein increased and the expression of ZO-1 protein decreased in IG 24 h,and there was no significant difference in RG.Compared with RG,the expression of Claudin-1 protein in IG 24 h increased and the expression of ZO-1 protein decreased.(4)RNA-seq analysis: compared with CG and RG,the genes related to synthesis,transport and regulation of milk components(lactoprotein,lactose,butterfat)of IG were significantly down-regulated,and energy-related genes were significantly down-regulated.The differential gene enrichment pathway NF-κB signal pathway and rapamycin target protein(mTOR)signal pathway were screened.The results of Westernblot verification showed that the level of IκB protein phosphorylation in IG and RG increased significantly,while the level of RG24 h rp S6 protein phosphorylation decreased.The above results suggest that the systemic inflammatory response induced by LPS can reduce food intake,increase the contents of PRL and decrease the contents of GH,destroy the balance of redox,lead to the shedding of mammary epithelial cells,damage tight junctions,activate NF-κB signal pathway,down-regulate the expression of lactation-related genes,and then inhibit lactation in mice.