Selenium Regulates The Mechanism Of JAKs/STATs-PI3K/Akt Signaling Axis Through LncRNA 802.1 To Inhibit Mammary Inflammatory Injury In Dairy Cows

Selenium(Se)is a trace element essential for the maintenance of health in animal organisms.It plays a key biological function in tissues and organs.Se is involved in the formation of 30 important selenoproteins in 25 mammalian species.These selenoproteins regulate the intracellular inflammatory signaling cascade to maintain the internal environment.Numerous feeding trials proved that the addition of appropriate amounts of Se to the basal diet of dairy cows enhances cellular immunity,and influences humoral immunoglobulins,improves growth performance,and significantly alleviates the extent of mammary inflammatory lesions.In recent years,the regulatory role of long non-coding RNAs(lncRNAs)is a focus.LncRNAs are widely involved in gene regulation,biological processes,and inflammatory and immune responses in a variety of diseases.Some studies have indicated that lncRNAs are involved in pathophysiological processes in animal mammary glands.Although early progress has been made in the study of the molecular basis of mammary inflammation in cows.The study of lncRNA transcriptional profiles in cows and mammary tissues is still not abundant and complete.LncRNAs regulate certain types of cytokines and their mechanisms in mammary inflammation in dairy cows need to be further explored and verified.Therefore,this study was established based on an inflammatory response model of mammary epithelial cells of dairy cows(MAC-T)cells treated with different concentrations of selenomethionine.LncRNA and m RNA high-throughput genomics sequencing means and bioinformatics analysis were applied.GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis showed that the NF-κB pathway,PI3K-Akt signaling pathway,and JAK-STAT signaling pathway in the control,LPS stimulation group,and Se+LPS group were co-enriched among the three groups.There were 14 identical lncRNAs were identified and noticed,among which lncRNA 802.1 was locked as the target lncRNA for this study cause of its significant expression difference and abundant target gene.The expression level of lncRNA 802.1 was verified by q PCR method,which was consistent with the trend of histological results.LncRNA 802.1 was significantly increased in the LPS-stimulated group,while the level in the Se-treated group was decreased,with a trend of a concentration gradient.q PCR and Western blot(WB)results showed that the expression levels of m RNA and protein expression of JAKs/STATs-PI3K/Akt pathway-related factors,such as JAK1,JAK2,STAT3,PI3 K,and Akt,were reduced in MAC-T cells after Se treatment compared to the LPS group.WB and immunofluorescence staining showed that Se addition significantly inhibited the phosphorylation levels of IκBα and NF-κB p65,and reduced the production of pro-inflammatory factors,resulting in decreasing the inflammatory response within MAC-T cells.To investigate whether Se regulates three differential express pathways through lncRNA802.1 and thus affects the inflammatory response in MAC-T cells.In this study,lncRNA 802.1knockdown experiments were performed in MAC-T cells.q PCR and ELISA were used to detect the m RNA and protein levels of inflammatory factors IL-1β,TNF-α,and IL-6.The results showed that pro-inflammatory factors were significantly reduced after lncRNA knockdown.q PCR and WB results showed that JAKs/STATs-PI3K/Akt pathway-related genes were significantly decreased,and the phosphorylation levels of IκBα and NF-κB p65 were suppressed.The above results proved that the knockdown of lncRNA802.1 could effectively inhibit the JAKs/STATs-PI3K/Akt pathway as well as the NF-κB pathway,resulting in the reduction of the expression of inflammatory factors.Considering the realistic conditions,economic factor,and experimental controllability,this study used mice for the animal regression model.Studies have proved that the mammary duct perfusion LPS method by mice can successfully establish a mammary inflammatory injury model,which is a reliable alternative model of mastitis in dairy cows.Therefore,40 female mice at 7d of parturition were divided equally into 4groups: the control group(a normal diet containing 0.18 mg/kg selenium),LPS stimulated group(normal diet containing 0.18 mg/kg selenium),high selenium group(a diet containing 0.6 mg/kg selenium),and high selenium +LPS group(a diet containing 0.6 mg/kg selenium)in this study.The mammary duct infusion lipopolysaccharide(LPS,0.2 mg/m L)method was used for the establishment of animal models of mastitis in mice.H&E staining of mouse mammary tissue sections showed severe inflammatory cell infiltration and disruption of the structural integrity of the glandular vesicles in the LPS group.While the reduced inflammatory infiltration in the mammary tissue of mice in HSe+LPS group.q PCR result showed that lncRNA802.1 was highly expressed in mammary tissues of mice in the LPS-stimulated group,and its level was reduced in mammary tissues of mice in the Se+LPS group.q PCR and ELISA results showed that the m RNA and protein expression related to the NF-κB pathway and JAKs/STATs-PI3K/Akt pathway were significantly lower in mammary tissues of mice fed Se-enriched grain than in the LPS group,which was consistent with the results of in vitro experiments.In conclusion,trace element Se could inhibit JAKs/STATs-PI3K/Akt pathway through lncRNA802.1 and inhibit IκBα and NF-κB p65 phosphorylation,thus alleviating LPS-induced inflammatory response in MAC-T cells.