Hesperidin ameliorates H_(2)O_(2)-induced bovine mammary epithelial cell oxidative stress via the Nrf2 signaling pathway

Background Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells(b MECs) exposed to oxidative stress have not been elucidated.Results In this study, we investigated the effects of hesperidin on H_(2)O_(2)-induced oxidative stress in b MECs and the underlying molecular mechanism. We found that hesperidin attenuated H_(2)O_(2)-induced cell damage by reducing reactive oxygen species(ROS) and malondialdehyde(MDA) levels, increasing catalase(CAT) activity, and improving cell proliferation and mitochondrial membrane potential. Moreover, hesperidin activated the Keap1/Nrf2/ARE signaling pathway by inducing the nuclear translocation of Nrf2 and the expression of its downstream genes NQO1 and HO-1, which are antioxidant enzymes involved in ROS scavenging and cellular redox balance. The protective effects of hesperidin were blocked by the Nrf2 inhibitor ML385, indicating that they were Nrf2 dependent.Conclusions Our results suggest that hesperidin could protect b MECs from oxidative stress injury by activating the Nrf2 signaling pathway, suggesting that hesperidin as a natural antioxidant has positive potential as a feed additive or plant drug to promote the health benefits of bovine mammary.

Sulforaphane prevents LPS‑induced inflammation by regulating the Nrf2‑mediated autophagy pathway in goat mammary epithelial cells and a mouse model of mastitis

Background Mastitis not only deteriorates the composition or quality of milk,but also damages the health and pro-ductivity of dairy goats.Sulforaphane(SFN)is a phytochemical isothiocyanate compound with various pharmacologi-cal effects such as anti-oxidant and anti-inflammatory.However,the effect of SFN on mastitis has yet to be elucidated.This study aimed to explore the anti-oxidant and anti-inflammatory effects and potential molecular mechanisms of SFN in lipopolysaccharide(LPS)-induced primary goat mammary epithelial cells(GMECs)and a mouse model of mastitis.Results In vitro,SFN downregulated the mRNA expression of inflammatory factors(tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand IL-6),inhibited the protein expression of inflammatory mediators(cyclooxygenase-2(COX2),and inducible nitric oxide synthase(iNOS))while suppressing nuclear factor kappa-B(NF-κB)activation in LPS-induced GMECs.Additionally,SFN exhibited an antioxidant effect by increasing Nrf2 expression and nuclear translocation,up-regulating antioxidant enzymes expression,and decreasing LPS-induced reactive oxygen species(ROS)produc-tion in GMECs.Furthermore,SFN pretreatment promoted the autophagy pathway,which was dependent on the increased Nrf2 level,and contributed significantly to the improved LPS-induced oxidative stress and inflammatory response.In vivo,SFN effectively alleviated histopathological lesions,suppressed the expression of inflammatory factors,enhanced immunohistochemistry staining of Nrf2,and amplified of LC3 puncta LPS-induced mastitis in mice.Mechanically,the in vitro and in vivo study showed that the anti-inflammatory and anti-oxidative stress effects of SFN were mediated by the Nrf2-mediated autophagy pathway in GMECs and a mouse model of mastitis.Conclusions These results indicate that the natural compound SFN has a preventive effect on LPS-induced inflam-mation through by regulating the Nrf2-mediated autophagy pathway in primary goat mammary epithelial cells and a mouse model of mastitis,which may improve prevention strategies for mastitis in dairy goats.

Primary Culture of Bovine Mammary Epithelial Cells

[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [ Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2 -4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [ Conclusion] Primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.

Optimization of Parameters of Exogenous Gene Mediated by Liposome to Transfect Yak Mammary Epithelial Cells in Vitro

[Objective] The aim of this study was to optimize conditions of exogenous gene mediated by liposome to transfect yak mammary epithelial cells in Vitro.[Method] Yak mammary epithelial cells were isolated and cultivated in Vitro by the methods of collagenase digestion and tissue adhesion.The expression of cytokeratin in yak mammary epithelial cell was detected by immunocytochemistry technique.With green fluorescence protein as the report gene,yak mammary epithelial cells were transfected with exogenous gene m...

Influence on Cellular Signal Transduction Pathway in Dairy Cow Mammary Gland Epithelial Cells by Galactopoietic Compound Isolated from Vaccariae segetalis

The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate （DBP） separated from Vaccaria segetalis on the expression of lactation signal transduction genes of mammary gland epithelial cells, including prlr, erα, akt1, socs2, pparγ and elf5, will be helpful to reveal the molecular mechanism. Western blot and qRT- PCR were used to study the change of prlr, erα, akt, socs2, pparγ, and elf5 expression at mRNA and protein level. Co- localization expression of prolactin receptor （PRLR） and estrogen receptor α （ERα） was observed by immunofluorescence; the expression changes of miRNAs （21, 125b, 143, and 195） and the secretion of β-casein and lactose were detected by qRT-PCR and RP-HPLC. The results showed that Vaccaria segetalis active compound had similar fuctions as estrogen and/or prolactin （PRL） in dairy cow mammary gland epithelial cells （DCMECs）, increased the expressions of prlr, erα, akt1, and elf5 genes, while repressed pparγ expressions. DBP promoted socs2 mRNA expression, but its protein expressions were repressed. Furthermore, both DBP and PRL could repress the expressions of miRNA-125b, miRNA-143 and miRNA- 195 in DCMECs. DBP could repress the expression of miRNA-21, while the influence of PRL on miRNA-21 was not certain. DBP could promote the lactation ability of DCMECs by regulating the ER and PRLR cellular signal transduction pathway.

Metabolic Regulation of Mammary Gland Epithelial Cells of Dairy Cow by Galactopoietic Compound Isolated from Vaccariae segetalis

In previous experiment, we isolated a compound dibutyl phthalate （DBP） from Vaccaria segetalis which had galactopoietic function on mammary gland epithelial cells of dairy cow （DCMECs）. In this experiment, we ascertained the metabolic regulation function of DBP on DCMECs. Many genes related to lactation including Stat5, AMPK, b-casein, Glut1, SREBP-1, PEPCK, and ACC were detected by real-time PCR. Furthermore, Stat5 and AMPK were detected by Western blot and immunofluorescence co-localization, respectively. The results showed that DBP stimulates the expression of Stat5 and p-Stat5, thus activates Stat5 cell signal transduction pathway and stimulates b-casein synthesis. DBP also raises the activities of Glut1 and AMPK to stimulate glucose uptake and glycometabolism and activates the expression of AMPK downstream target genes PEPCK and ACC and expression of SREBP-1 to stimulate milk fat synthesis. In addition, the activities of HK, G-6-PDH, ICDH, ATPase, and energy charges were stimulated by DBP to increase the energy metabolism level of DCMECs. The results showed DBP stimulates energy metabolism related to galactopoietic function in DCMECs.

miR-25 modulates triacylglycerol and lipid accumulation in goat mammary epithelial cells by repressing PGC-1beta

Background: The goat(Caprahircus) is one of the most important livestock animals. Goat milk fat is an important component in the nutritional quality of goat milk. Growing evidence points to the critical roles of microRNAs(miRNAs) in lipid metabolism.Results: Using a highly sensitive method of S-poly(T) plus for miRNAs detection, we analyze the expression patterns of 715 miRNAs in goat mammary gland tissues at different stages of lactation. We observed that miR-25 expression had an inverse relationship with milk production. Overexpression of miR-25 significantly repressed triacylglycerol synthesis and lipid droplet accumulation. To explore the regulatory mechanism of miR-25 in milk lipid metabolism,we analyzed its putative target genes with bioinformatics analysis followed by 3′-UTR assays. Peroxisome proliferative activated receptor gamma coactivator 1 beta(PGC-1 beta), a key regulator of lipogenics was identified as a direct target of miR-25 with three specific sites within its 3′-UTR. In addition, miR-25 mimics in goat mammary epithelial cells reduced the expressions of genes involved in lipid metabolism.Conclusions: Taken together, our results show miR-25 is potentially involved in lipid metabolism and we reveal the function of the miR-25/PGC-1 beta regulatory axis during lactation.

Effect of Semen vaccariae and Taraxacu mogono on Cell Adhesion of Bovine Mammary Epithelial Cells

The aim of this study is to reveal the regulation mechanism of the effect of Semen vaccariae and Taraxacu mogono on the cell-cell adhersion molecule, E-cadherin and β-catenin on the proliferation role and secretion function of bovine mammary epithelial cells cultured in vitro. Firstly, the epithelial character of bovine mammary epithelial cells was authenticated using immunofluorescence, then the cell grow curve was observed and investigated after S. vaccariae and T. mogono treatment. On the effect of S. vaccariae and T. mogono, cell adhesion molecules E-cadherin, β-catenin and CycinD1 mRNA and protein were detected by qRT-PCR and Western blotting, respectively. The results showed that the cellular keratin 18 expressed positively and proliferfated vigorously after S. vaccariae and T. mogono treament. The mRNA and protein levels of E-cadherin and CycinD1 were remarkably higher （P〈0.05） in 36 h after S. vaccariae and T. mogono treatment. The cell proliferation at 36 h was increased significantly （P〈0.05）. In conclusion, S. vaccariae and T. mogono have a positive impact on the cell proliferation and an effect on the adhesion molecules E-cadherin, β-catenin and CycinD1 in the Wnt signaling pathway.

The protective roles of tea tree oil extracts in bovine mammary epithelial cells and polymorphonuclear leukocytes

Background: Tea tree oil(TTO) plays an important role in antibacterial activity and alleviating the inflammatory responses. Bovine mammary epithelium and polymorphonuclear leukocytes(PMNL) can actively respond to bovine mastitis infection. However, regulatory effects of TTO extracts on the innate immune response of bovine mammary epithelial cells(BMECs) and PMNL remain not reported. Therefore, aim of the study was to evaluate the effects of TTO extracts on the m RNA levels of the genes involved in the innate immune response of BMECs and PMNL.Results: Our results demonstrated that addition of 0.025% and 0.05% TTO increased the proliferation of BMECs, and significantly enhanced(P < 0.05) the viability of BMECs exposed to Staphylococcus aureus(S. aureus). An inhibitory effect was observed against the growth of S. aureus by TTO incubation. The 0.05% TTO reduced S. aureus biofilm formation, association and invasion of S. aureus to BMECs, and changed the morphological and structural features of S. aureus. The proinflammatory cytokines IL-1β, IL-6, and TNF-α were decreased(P < 0.001) by the incubation of TTO. Interestingly, the expression of IL-8 known for PMNL chemotactic function was elevated(P < 0.05) by 0.05%TTO treatment. Consistently, 0.05% TTO increased the migration of PMNL in S. aureus-exposed BMECs when compared with S. aureus treatment alone(P < 0.05). In addition, PMNL incubated with 0.05% TTO decreased the levels of NFKB inhibitor alpha(NFKBIA) and TNF-α.Conclusions: Our results indicate that use of TTO can relieve the BMECs pro-inflammatory response caused by S.aureus and promote the migration of PMNL to mount the innate immune responses, and it may be novel strategy for the treatment of bovine mastitis caused by S. aureus.

Knockout of butyrophilin subfamily 1 member A1(BTN1A1) alters lipid droplet formation and phospholipid composition in bovine mammary epithelial cells

Background: Milk lipids originate from cytoplasmic lipid droplets(LD) that are synthesized and secreted from mammary epithelial cells by a unique membrane-envelopment process. Butyrophilin 1 A1(BTN1 A1) is one of the membrane proteins that surrounds LD, but its role in bovine mammary lipid droplet synthesis and secretion is not well known.Methods: The objective was to knockout BTN1 A1 in bovine mammary epithelial cells(BMEC) via the CRISPR/Cas9 system and evaluate LD formation, abundance of lipogenic enzymes, and content of cell membrane phospholipid(PL) species. Average LD diameter was determined via Oil Red O staining, and profiling of cell membrane phospholipid species via liquid chromatography-tandem mass spectrometry(LC-MS/MS).Results: Lentivirus-mediated infection of the Cas9/sg RNA expression vector into BMEC resulted in production of a homozygous clone BTN1 A1^((-/-)). The LD size and content decreased following BTN1 A1 gene knockout. The m RNA abundance of fatty acid synthase(FASN) and peroxisome proliferator-activated receptor-gamma(PPARG) was downregulated in the BTN1 A1^((-/-))clone. Subcellular analyses indicated that BTN1 A1 and LD were co-localized in the cytoplasm. BTN1 A1 gene knockout increased the percentage of phosphatidylethanolamine(PE) and decreased phosphatidylcholine(PC), which resulted in a lower PC/PE ratio.Conclusions: Results suggest that BTN1 A1 plays an important role in regulating LD synthesis via a mechanism involving membrane phospholipid composition.

Transcription factor EB(TFEB)-mediated autophagy protects bovine mammary epithelial cells against H_(2)O_(2)-induced oxidative damage in vitro

Background:Bovine mammary epithelial cells after calving undergo serious metabolic challenges and oxidative stress both of which could compromise autophagy.Transcription factor EB(TFEB)-mediated autophagy is an important cytoprotective mechanism against oxidative stress.However,effects of TFEB-mediated autophagy on the oxidative stress of bovine mammary epithelial cells remain unknown.Therefore,the main aim of the study was to investigate the role of TFEB-mediated autophagy in bovine mammary epithelial cells experiencing oxidative stress.Results:H_(2)O_(2) challenge of the bovine mammary epithelial cell MAC-T increased protein abundance of LC3-II,increased number of autophagosomes and autolysosomes while decreased protein abundance of p62.Inhibition of autophagy via bafilomycin A1 aggravated H_(2)O_(2)-induced reactive oxygen species(ROS)accumulation and apoptosis in MAC-T cells.Furthermore,H_(2)O_(2) treatment triggered the translocation of TFEB into the nucleus.Knockdown of TFEB by siRNA reversed the effect of H_(2)O_(2) on protein abundance of LC3-II and p62 as well as the number of autophagosomes and autolysosomes.Overexpression of TFEB activated autophagy and attenuated H_(2)O_(2)-induced ROS accumulation.Furthermore,TFEB overexpression attenuated H_(2)O_(2)-induced apoptosis by downregulating the caspase apoptotic pathway.Conclusions:Our results indicate that activation of TFEB mediated autophagy alleviates H_(2)O_(2)-induced oxidative damage by reducing ROS accumulation and inhibiting caspase-dependent apoptosis.

Establishment of Immortalized Cow Mammary Epithelial Cells Expressing Both h TERT and SV40 T

Vectors of pcDNA3.1-hTERT and pcDNA 3. 1-SV40 T were established. After linearization, they were cotransfected to mammary epithelial cells of Holstein cow, in order to research on the role of hTERT and SV40 T in immortalized mammary epithelial cells in vitro. Both PT-PCR and immunohistochemical as- says of cells were carried out. Results showed that the expression of hTERT and SV40 T could effectively prolong the culture time in vitro of mammary epithelial cells, and enhance the cell passage number. The obtained cell line could be expressed normally, indicating that the in vitro cultured mammary epithelial cells expressing both hTERT and SV40 T could effectively prolong cell llfe without affecting the characteristics of mammary cells.

Bta-miR-34b controls milk fat biosynthesis via the Akt/mTOR signaling pathway by targeting RAI14 in bovine mammary epithelial cells

Background:The biosynthesis of milk fat affects both the technological properties and organoleptic quality of milk and dairy products.MicroRNAs(miRNAs)are endogenous small non-coding RNAs that inhibit the expression of their mRNA targets and are involved in downstream signaling pathways that control several biological processes,including milk fat synthesis.miR-34b is a member of the miR-34 miRNA cluster,which is differentially expressed in the mammary gland tissue of dairy cows during lactation and dry periods.Previous studies have indicated miR-34b is a potential candidate gene that plays a decisive role in regulating milk fat synthesis;therefore,it is important to focus on miR-34b and investigate its regulatory effect on the biosynthesis of milk fat in bovine mammary epithelial cells(BMECs).Results:In this study,elevated miR-34b levels reduced milk fat synthesis,upregulated 1,999 genes,and downregulated 2,009 genes in BMECs.Moreover,Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis of differentially expressed genes suggested that miR-34b may play an inhibitory role in milk fat synthesis via the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway by reducing phosphorylation levels.Notably,the mTOR activator MHY1485 rescued the inhibitory effect of miR-34b.Furthermore,we demonstrated that retinoic acid-induced protein 14(RAI14)is a target of miR-34b via TargetScan and immunofluorescence assays.RAI14 mRNA and protein levels were significantly decreased by the miR-34b mimic and increased by the miR-34b inhibitor.Moreover,the reduction in RAI14 levels led to the inhibition of the Akt/mTOR signaling pathway.Conclusions:Overall,our results identified a miR-34b-RAI14-Akt/mTOR regulatory network,while also providing a theoretical basis for the molecular breeding of dairy cows.

Tobacco-specific Carcinogen 4-(Methylnitrosoamino)-1-(3-pyridyl)-1-butanone(NNK) Activating ERK1/2 MAP Kinases and Stimulating Proliferation of Human Mammary Epithelial Cells

Cigarette smoking is correlated with the development of various cancers. 4- （Methylnitresoamino） -1- （3-pyridyl） - 1-butanone（NNK） is one of the major tobacco-specific carcinogens in the cigarette smoke, which increases the risk of breast cancer. In the present study, it was demonstrated that NNK rapidly activated ERK1 and ERK2 MAP kinases in human normal mammary epithelial cells. It was found that there are two different routes for the activation of ERK1/2 with NNK. One is from nicotinic receptor nAchR to MEK1/2, and the other is from tyrosine kinase containing receptor to MEK1/2. The tobacco-specific carcinogen NNK shows a strong proliferative effect on normal human mammary epithelial cells and cancer mammary epithelial cells.

Construction of Antibacterial Peptide CecropinB Eukaryotic Recombinant Vector and Its Expression in Dairy Goat Mammary Gland Epithelial Cells

To investigate the expression of antibacterial peptide CecropinB cDNA in dairy goat mammary gland epithelial cells,the CecropinB gene was cloned and was inserted into a eukaryotic vector pECFP-C1 to construct the recombinant plasmid pECFP-B by genetic engineering technique.Recombinant plasmid pECFP-B was transfected into dairy goat mammary gland epithelial to detect the bactericidal activity of CecropinB.The expression of CecropinB was also detected.The result of RT-PCR demonstrated CecropinB gene was expressed in transfected cells.CecropinB recombinant plasmid DNA was injected into udders and CecropinB was expressed in mammary gland,exhibiting bactericidal activity to Staphylococcus aureus in vivo experiments.

Stable Expression of Antibacterial Peptide CecropinB in Dairy Goat Mammary Gland Epithelial Cells

The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.

Impact of let-7g on Proliferation and Lactation of Mouse Mammary Epithelial Cells

let-7g, a member of the let-7 family, regulates gene expression at the post-transcriptional level. The study explored a series of biological effects of mouse mammary epithelial cells that let-7g was produced. The differential expression of let-7g was detected by qRT-PCR in different developmental stages of the mouse mammary gland, let-7g expression and impact of let-7g on mouse mammary epithelial cells were analyzed by CASY-technology, qRT-PCR, Western blotting and HPLC inhibited let-7g expression of mouse mammary epithelial ceils through gene silencing. The results showed that qRT-PCR identified let-7g as being down-regulated in mouse mammary epithelial cells after it was inhibited. Mouse mammary epithelial cells with low expression of let-7g displayed higher expression of TGFβR I protein than those with high expression of let-7g, suggesting that low let-7g expression contributed to TGFβR I over-expression. Finally, the expression of let-7g was down-regulated, which significantly enhanced the proliferation of mouse mammary epithelial cells, and increased expression of β-Casein. The data indicated that let-7g could negatively regulate the expression of target Tgfbrl by complementary combination in mouse mammary epithelial cells, and then regulate the cell proliferation and expression of β-Casein by suppressing the TGFβR I expression.

Effect of Different Concentrations of Lipopolysaccharide on Expression of NF-κB and LAP in Mammary Epithelial Cells of Dairy Cow

The paper was to study the effects of different concentrations of lipopelysaccharide （LPS） on expression of nuclear factor Kappa B （NF-κB） and lingual antimicrobial peptide （LAP） gene in mammary epithelial ceils of dairy cow. The mammary epithelial ceils of dairy cow were stimulated by different concentrations （50, 100,200,400 and 800 ng/mL） of LPS. The total RNA of cells was extracted after stimulation for 2, 4, 8, 16, 24, 48 and 72 h, respectively, and the mRNA expression levels of NF-κB P65 and LAP were evaluated by real-time quantitative PCR. The results showed that the expression of NF-κB P65 and LAP mRNA treated with 400 ng/mL LPS for 72 h were the highest compared to the control group （ P 〈0.01 ）. The result confirmed that the expression activity of NF-κB was enhanced in inflammatory effects of inammary epithelial cells induced by LPS, which regulated the expression of defense gene LAP, with certain dose and time effects.

MiR-140 downregulates fatty acid synthesis by targeting transforming growth factor alpha(TGFA)in bovine mammary epithelial cells

Fat is an indispensable nutrient and basic metabolite for sustaining life,and milk is particularly rich in fatty acids,including a variety of saturated and unsaturated fatty acids.MicroRNA(miRNA)and mRNA play an important role in the regulation of milk fat metabolism in mammary gland tissue.It has been shown that lipid metabolism has a complex transcriptional regulation,but the mechanism by which milk fat synthesis is regulated through miRNA–mRNA interactions is poorly understood.In this study,we performed transcriptome sequencing with bovine mammary gland tissue in the late lactation(270 and 315 days after parturition)to identify the key gene that regulating milk fat metabolism.A total of 1207 differentially coexpressed genes were selected,828 upregulated genes and 379 downregulated genes were identified.The transforming growth factor alpha(TGFA)gene was selected as the target gene,and luciferase reporter assay,Western blotting and q RT-PCR were used for further study.The results demonstrated that miR-140 was an upstream regulator of TGFA,and miR-140 could inhibit(P<0.01)unsaturated fatty acid and triglyceride(TAGs)production in bovine mammary epithelial cells(BMECs).In contrast,TGFA promoted(P<0.01)unsaturated fatty acid and TAG production.Rescue experiments further indicated the mi R-140/TGFA regulatory mechanism.Taken together,these results suggest that the mi R-140/TGFA pathway can inhibit(P<0.01)milk fat metabolism and improve milk quality by genetic means.

NH4Cl promotes apoptosis and inflammation in bovine mammary epithelial cells via the circ02771/miR-194b/TGIF1 axis

Excess ammonia(NH_(3))in the circulation of dairy animals can reduce animal health and the quality of products for human consumption.To develop effective prevention and treatment methods,it is essential to examine the molecular mechanisms through which excess NH_(3) may affect the mammary gland.The present study used bovine mammary epithelial cells(BMECs)to evaluate the effects of exogenous NH_(4)Cl on the abundance of circular RNAs(circRNAs)using high-throughput sequencing.Among the identified circRNAs,circ02771 was the most significantly upregulated by exogenous NH_(4)Cl(P<0.05),with a fold change of 4.12.The results of the apoptosis and proliferation assays,transmission electron microscopy,H&E staining,and immunohistochemistry revealed that circ02771 increased apoptosis and inflammation.A double luciferase reporter assay revealed that circ02771 targeted miR-194b,and the overexpression of circ02771(pcDNA-circ02771)reduced(P<0.05)the expression of miR-194b and led to apoptosis and inflammation.Circ02771 also enhanced the expression of transforming growth factor beta-induced factor homeobox 1(TGIF1),which is a target gene of miR-194b.Overall,this study suggests that the circ02771/miR-194b/TGIF1 axis plays a role in mediating the effects of NH_(4)Cl on BMECs.Therefore,this axis provides a novel target to help control hazards within the mammary gland from high circulating NH_(4)Cl levels.