Role And Mechanism Of UFL1 In Lactation Physiology And Mastitis In Mouse Mammary Gland

Ubiquitin-fold modifier 1 specific ligase 1(UFL1,UFM-specific ligase 1)is an important member of UFM1(Ubiquitin-fold modifier 1)conjugation system,which is expressed in many tissues and organs such as the heart,liver,small intestine,and pancreas.In addition,UFL1 is a key regulatory protein required for diverse physiological processes including: DNA damage,apoptosis,autophagy and so on.Studies from several types of mammal cells with strong metabolism found that UFL1 knockout activates celluar disorders,and then damage body functions.Studies have suggested that UFL1 can affect the level of pancreatin secretion via regulate the prancreatin β cell.It is suggested that UFL1 is a potential protein to affect cell secretion.Our previous studies that the inflammatory response of dairy cow mammary epithelial cells is accompanied by an increase in UFL1 expression.Studies have confirmed that UFL1 is expressed in the human mammary gland However,the relationship between UFL1 and milk fat,milk protein synthesis,and UFL1 and inflammation is still unclear.Based on this,UFL1 knockout mice and mouse mammary epithelial cells(HC11 cells line)were used as the research materials to understand the expression patterns of UFL1 in mouse mammary tissues.Knockout UFL1 or overexpression at the mice and cells levels were used to explore the role of UFL1 in the process of mouse lactation;and analyzed UFL1 as a target protein that regulates LPS-induced mouse mastitis and its potential signaling pathways.Through the role and mechanism of UFL1 in the lactation process of mouse mammary glands and the treatment of mastitis have been preliminarily clarified.This result enriches the molecular regulatory network that regulates lactation and mastitis treatment.It can provide new target theoretical reserves and scientific and technical support for improving the quality of lactation,breast inflammation treatment and new target drug design.The main results are as follows:(1)UFL1 is expressed in mouse mammary epithelial cellsThe localization of UFL1 were anayzed in mice mammary gland from postanatal 6-7days.The results of immunohistochemistry showed that UFL1 was strongly expressed in mouse mammary tissue luminal epithelial cells(brown location),and almost not expressed in adipose tissue.In vitro: immunofluorescence analysis suggested that UFL1 is strongly expressed(localized in red)in the cytoplasm,while weakly localized in the nucleus.After the HC11 nuclear protein and the plasmin were completely separated,it was found that UFL1 was highly expressed in the cytoplasmic protein,but weakly or even not expressed in the nucleus.(2)Effects of UFL1 on the morphological structure,body mass index and mammary index of miceUFL1 KO mice were used for detected whether UFL1 deficiency alteraed mammary gland weight and morphology.According to HE analysis,there is no difference in the histomorphology of mammary gland between UFL1 knockout mice and wild-type mice,and both contain a lot of acinar tissue and adipose tissue.I In addition,between UFL1 knockout mice and wild-type mice,the mouse body weight and mammary gland index weight(the ratio of the fourth pair of mammary gland weight to the mouse body weight)did not change significantly.(3)Effect of UFL1 on the level of milk fat milk and protein synthesis related genesReducing endogenous UFL1 significantly up-regulated the expression level of triglyceride in UFL1 KO mice.Triglyceride secretion is inseparable from the regulation of milk fat synthesis related genes(ACACA and FASN).When the expression of UFL1 konckout,the expression of milk fat synthesis(ACACA and FASN)will increase significantly,and decrease the expression of CSN2.HC11 cells were tranfectd with si UFL1.Results have found that UFL1 knockdown were no affects the level of TG.Western blot has showed that the expression of CSN2 protein in the si UFL1 group was significantly lower than that in the Control group,but the protein expression of ACACA and FASN increased significantly.In summary,knockout of UFL1 promotes the increase in triglyceride levels and the expression of milk fat synthesis-related genes,but inhibits the decrease in the expression of milk protein synthesis-related genes.UFL1 plays a key role in the regulation of milk fat and protein synthesis in the mammary gland.1.Effects of UFL1 on the changes of milk fat and milk protein related genes in mouse mammary glands2.Study on the mechanism of UFL1 affecting milk fat and milk protein-related regulatory genes(1)Effect of UFL1 on apoptosis of mouse mammary gland cellsKnockout of UFL1 to promote cell apoptosis factor(BAX to BCL-2 ratio and CleavedCaspase3)protein expression increased significantly.This result suggested that apoptosis was activeated.HC11 cells were transfected with si UFL1 also activated apoptosis.In addition,we also used flow cytometry and TUNEL test technology to analyze cell apoptosis.After HC11 knocked out UFL1 expression,the apoptotic cell ratio was significantly up-regulated.The CCK8 kit was used to detect the effect of si UFL1 transfection on the viability of HC11 cells in different time periods.It was found that si UFL1 transfection had no significant effect on HC11 cells for 24 h,but cell viability was inhibited at 48 h and 72 h after transfection,and the inhibitory effect increased with time.(2)UFL1 affects the level of milk fat and milk protein related genes through JNK activityStudies have shown that JNK is an important factor affecting the growth and development of mammary glands,and UFL1 also regulates a number of physiological processes by affecting the activity of JNK.Maybe JNK is an important target of UFL1 affecting the synthesis of milk fat and milk protein.First,we analyzed the effect of UFL1 on JNK activity in mouse mammary glands.Whether UFL1 knockout mice or HC11 cells transfected with si UFL1: there is no significant effect on the expression of JNK protein after reducing endogenous UFL1,the expression of p-JNK protein decreases,and the ratio of pJNK to JNK protein decreases significantly.Similar results were obtained by analyzing the activity of JNK by ELISA.Based on the previous results,we believe that the UFL1-JNK pathway is likely to be an important pathway to regulate the changes in milk fat and milk protein synthesis-related genes.SP600125 is an inhibitor of JNK and used for inhibited the active of JNK in HC11 cells.The groupings were as follows: Control group,DMSO group,SP600125 group and si UFL1+SP600125 group.The results of immunoblotting showed that compared with the Control group and the DMSO group,when the JNK activity was inhibited(SP600125 group)CSN2 protein expression was significantly inhibited,and the CSN2 protein expression was lower in the si UFL1+SP600125 group;but after JNK was inhibited,the ACACA and FASN protein levels has opposite effects.At the same time,compared with the Control group,ACACA and FASN in the si UFL1+ SP600125 group increased.3.The role of UFL1 in LPS-induced mastitis in mice mammary gland(1)LPS-induced mammary gland inflammation reponse promotes increased UFL1 expression in miceLPS is the main component of the gram-negative bacteria wall and is widely used in basic research on inducing inflammation response in mammary.LPS were injected into the fourth pair of mammary glands of mice 5-7 days postpartum can specifically cause mastitis in mice.Accompanied by inflammation,the expression of UFL1 increased significantly.In addition,HC11 cells cultured were treated with LPS at a concentration of 800 ng/m L to induce the inflammation reponse in mammary epithelial cells,and the expression level of UFL1 was analyzed.The results showed that the protein level and RNA level of UFL1 were increased followed LPS-treated.The RNA level of UFM1 members were also increacred after LPS-treated.(2)Effect of UFL1 on the expression levels of inflammatory mediators and inflammatory cytokines in LPS-induced mammary gland inflammation in miceIn order to further explore the role of UFL1 in mice mastitis,we used UFL1 knockdown mice and HC11 cells as the research material in this experiment.UFL1 knockout mice are grouped into: WT group,LPS group,LPS+UFL1 KO group and UFL1 KO group.The results show that UFL1 is closely related to the expression of pro-inflammatory mediators and proinflammatory cytokines in LPS-induced mastitis.Knockout UFL1 increased the expression of pro-inflammatory mediators(i NOS,MDA,SOD and GSH-PX)and pro-inflammatory cytokines(IL1β,IL6 and TNFα)induced by LPS.The in vitro experiments are grouped as follows: groups are as follows: Control group,LPS group,LPS+si UFL1 group,LPS+oe UFL1 group,si UFL1 group and oe UFL1 group.Transfection of si UFL1 aggravated the secretion of pro-inflammatory mediators(i NOS,MDA,SOD and GSH-PX)and pro-inflammatory cytokines(IL1β,TL6 and TNFα)caused by LPS.However,the UFL1 overexpression reduces the inflammatory responses caused by LPS.In summary,UFL1 can affect the process of inflammatory immune response by regulated the expression of pro-inflammatory mediators and cytokines in LPS-induced mammary gland inflammation response in mice.4.The Mechanism of UFL1 in LPS-induced Inflammatory Injury in Mouse Mammary Gland(1)Effect of UFL1 on inflammtation injury in mice mastitis induced by LPSMastitis is a complex and dynamic immune response process that is regulated by a variety of factors.It is often accompanied by inflammatory damage caused by the inflammatory response in the mammary gland.Mastitis induced by LPS often causes inflammatory damage to the health of mammary gland due to its intensity and duration.Same as the experimental grouping in the previous chapter.Knockout of UFL1 significantly promotes LPS-induced endoplasmic reticulum stress and activates the UPR response.In addition,reducing endogenous UFL1 increased LPS-induced LC3 expression,but there was no difference in p62 expression.This expriment is consistent with the third experiment HC11 cell test grouping.UFL1 knockdown can enhance the LPS-induced endoplasmic reticulum stress.After overexpression of UFL1,IRE protein can alleviate the occurrence of endoplasmic reticulum stress.At the same time,UFL1 knockdown promoted the down-regulation of p62 protein expression induced by LPS,but there was no difference change in LC3 protein.(2)Effect of UFL1 on the activity of NF-κB in mice mastitis induced by LPSIn order to clarify the mechanism of UFL1 on the NF-κB signaling pathway of LPSinduced inflammatory injury in mouse mammary glands.We tested the expression of members of the NF-κB signaling pathway.The results showed that the decrease of endogenous UFL1 aggravated the LPS-induced phosphorylated NF-κB activation.In addition,phosphorylated p-IKK-α/β and p-IκB-α were also significantly up-regulated.In vitro results confirmed that UFL1 has an effect on LPS-induced NF-κB in mouse mastitis tissues.In HC11 cells,UFL1 increased LPS-induced NF-κB phosphorylation and nuclear translocation,IKK-α/β,IκB-α phosphorylation expression level also increased.UFL1 knockdown further aggravated the activation of NF-κB signaling pathway,but overexpression of UFL1 significantly blocked the activation of NF-κB signaling pathway induced by LPS.(3)UFL1 affects the level of inflammatory cytokines in mice mastitis via NF-κB signaling pathwayPDTC is an effective inhibitor of the NF-κB signaling pathway,which can significantly inhibit the activity of NF-κB,and is widely used in a number of basic researches related to inflammation.PDTC was used to inhibit the activity of NF-κB in HC11 cells,and the changes in the expression levels of inflammatory factors were analyzed by ELISA.The results showed that blockade of NF-κB signaling pathway can effectively alleviate UFL1 involvement in the increase of inflammatory factors in LPS-induced mastitis in mice.The above results indicate that UFL1 regulates LPS-induced mastitis in mice through the NF-κB signaling pathway.