Schisandrin B inhibits inflammation and ferroptosis in S.aureus-induced mastitis through regulating SIRT1/p53/SLC7A11 signaling pathway.

Mastitis, one of the most significant problems in women, is commonly caused by pathogens, especially Staphylococcus aureus (S.aureus). Schisandrin B (SCB), the main abundant derivatives from Schisandra chinensis, has been proven to have the ability to inhibiting inflammation and bacteria. However, few relevant researches systematically illustrate the role SCB in the treatment of mastitis. The aim of the present study is to demonstrate the mechanism that SCB functions in reducing pathological injury to the mammary gland in treating S.aureus-induced mastitis. H&E staining was used to identify pathological changes and injuries in mastitis. The levels of cytokines associated with inflammation were detected by ELISA. Key signals relevant to ferroptosis and Nrf2 signaling pathway were tested by western blot analysis and iron assay kit. Compared with the control group, inflammation-associated factors, such as IL-1ß, TNF-α, MPO activity, increased significantly in S. aureus-treated mice. However, these changes were inhibited by SCB. Ferroptosis-associated factors Fe2+ and MDA increased significantly, and GSH, GPX4 and ferritin expression decreased markedly in S. aureus-treated mice. SCB treatment could attenuate S.aureus-induced ferroptosis. Furthermore, SCB increase SIRT1 and SLC7A11 expression and down-regulated p53 expression and NF-κB activation. In conclusion, SCB alleviates S.aureus-induced mastitis via up-regulating SIRT1/p53/SLC7A11 signaling pathway, attenuating the activation of inflammation-associated cytokines and ferroptosis in the mammary gland tissues.

In vivo and in vitro anti-inflammation of Rhapontici Radix extract on mastitis via TMEM59 and GPR161.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhapontici Radix ethanol extract (RRE) is derived from the dried root of Rhaponticum uniflorum (L.) DC belonging to the Asteraceae family. RRE exhibits significant anti-inflammatory and antioxidant properties; however, the potential of RRE in mastitis treatment requires further investigation. AIM OF THIS STUDY: This research was performed to examine the protective properties of RRE against mastitis and the mechanisms underlying the effects of RRE. MATERIAL AND METHODS: RRE components were analyzed by HPLC-MS/MS and DPPH methods. Isochlorogenic acid B (ICAB) was obtained commercially. MTT assay was utilized to assess RRE or ICAB cytotoxicity in bovine mammary alveolar (MAC-T) cells. Immunohistochemistry were used to investigate the pathological alterations in mammary tissue. The protein levels of inflammatory cytokines and mediators were analyzed using ELISA, and the expression of MAPK and NF-κB signaling pathways, as well as p65 nuclear translocation, were analyzed through Western blotting and immunofluorescence techniques, respectively. Target proteins of RRE were screened by RNA-seq and tandem mass tag analyses. Protein interaction was revealed and confirmed using co-immunoprecipitation and CRISPR/Cas9-based knockdown and overexpression of target genes. RESULTS: ICAB was revealed as one of the main components in RRE, and it was responsible for 84.33% of RRE radical scavenging activity. Both RRE and ICAB mitigated the infiltration of T lymphocytes in the mammary glands of mice, leading to decreased levels of inflammatory mediators (COX-2 and iNOS) and cytokines (TNF-α, IL-6, and IL-1ß) in lipopolysaccharide (LPS)-induced MAC-T cells. Furthermore, RRE and ICAB suppressed the LPS-induced phosphorylation of NF-κB inhibitor and p65, thereby impeding p65 nuclear translocation in mouse mammary glands and MAC-T cells. In addition, RRE and ICAB attenuated the LPS-triggered activation of c-Jun N-terminal kinase 1/2, p38, and extracellular regulated protein kinase 1/2. Importantly, co-treated with LPS and ICAB in MAC-T cells, an upregulation of G-protein coupled receptor 161 (GPR161) and transmembrane protein 59 (TMEM59) was observed; the interact between TMEM59 and was found, leading to inhibition of NF-κB activity and inflammatory cytokine production. CONCLUSION: ICAB is a prominent antioxidant in RRE. RRE and ICAB reduce mammary inflammation via MAPK and NF-κB pathways and the interaction between TMEM59 and GPR161 mediates the control of ICAB in NF-κB signaling.

Forsythiaside A attenuates lipopolysaccharide-induced mouse mastitis by activating autophagy and regulating gut microbiota and metabolism.

Mastitis is an inflammatory disease of the mammary gland with a high incidence in lactating animals, significantly impacting their health and breastfeeding. Moreover, mastitis adversely affects milk quality and yield, resulting in substantial economic losses for the dairy farming industry. Forsythiaside A (FTA), a phenylethanol glycoside analog extracted from Forsythia, exhibits notable anti-inflammatory and antioxidant properties. However, its protective effects and specific mechanisms against mastitis remain unclear. In this study, a lipopolysaccharide (LPS)-induced mouse mastitis model was used to investigate the protective effect of FTA on LPS-induced mastitis and its potential mechanism using histological assays, Western blot, qRT-PCR, FITC-albumin permeability test, 16s rRNA gene sequencing analysis and non-targeted metabolomics assays to investigate the protective effect of FTA on LPS-induced mastitis model and its potential mechanism. The results demonstrated that FTA significantly mitigated LPS-induced mouse mastitis by reducing inflammation and apoptosis levels, modulating the PI3K/AKT/mTOR signaling pathways, inducing autophagy, and enhancing antioxidant capacity and the expression of tight junction proteins. Furthermore, FTA increased the abundance of beneficial microbiota while decreasing the levels of harmful microbiota in mice, thus counteracting the gut microbiota disruption induced by LPS stimulation. Intestinal metabolomics analysis revealed that FTA primarily regulated LPS-induced metabolite alterations through key metabolic pathways, such as tryptophan metabolism. This study confirms the anti-inflammatory and antioxidant effects of FTA on mouse mastitis, which are associated with key metabolic pathways, including the restoration of gut microbiota balance and the regulation of tryptophan metabolism. These findings provide a novel foundation for the treatment and prevention of mammalian mastitis using FTA.

Effect and mechanism of Prunella vulgaris L. extract on alleviating lipopolysaccharide-induced acute mastitis in protecting the blood-milk barrier and reducing inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: According to ancient literature, Prunella vulgaris L. (P vulgaris) alleviates mastitis and has been used in China for many years; however, there are no relevant reports that confirm this or the mechanism of its efficacy. AIM OF THE STUDY: To explore the anti-acute mastitis effect and potential mechanism of P vulgaris extract. MATERIALS AND METHODS: First, the active ingredients and targets of P vulgaris against mastitis were predicted using network pharmacology. Next, the relevant active ingredients were enriched using macroporous resins and verified using UV and UPLC-Q-TOF-MS/MS. Lastly, a mouse model of acute mastitis was established by injecting lipopolysaccharides into the mammary gland and administering P vulgaris extract by oral gavage. The pathological changes in mammary tissue were observed by HE staining. Serum and tissue inflammatory factors were measured by ELISA method. MPO activity in mammary tissue was measured using colorimetry and MPO expression was detected by immunohistochemistry. The expression of tight junction proteins (ZO-1, claudin-3, and occludin) in mammary tissue was detected by immunofluorescence and Western blot. iNOS and COX-2 in mammary tissue were detected by Western blot. MAPK pathway and NF-κB pathway related proteins were also detected by Western blot. RESULTS: Network pharmacology predicted that phenolic acids and flavonoids in P vulgaris had anti-mastitis effects. The contents of total flavonoids and total phenolic acids in P vulgaris extract were 64.5% and 29.4%, respectively. UPLC-Q-TOF-MS/MS confirmed that P vulgaris extract contained phenolic acids and flavonoids. The results of animal experiments showed that P vulgaris extract reduced lipopolysaccharide-induced inflammatory edema, inflammatory cell infiltration, and interstitial congestion of mammary tissue. It also reduced the levels of serum and tissue inflammatory factors TNF-α, IL-6, and IL-1ß, and inhibited the activation of MPO. Furthermore, it downregulated the expression of MAPK and NF-κB pathway-related proteins. The expressions of ZO-1, occludin, and claudin-3 in mammary gland tissues were upregulated. CONCLUSIONS: P vulgaris extract can maintain the integrity of mammary connective tissue and reduce its inflammatory response to prevent acute mastitis. Its mechanism probably involves regulating NF-κB and MAPK pathways.

Hesperetin regulates the intestinal flora and inhibits the TLR4/NF-κB signaling axis to protect the blood-milk barrier and prevent mastitis.

The World Health Organization recommends breastfeeding for 6 months, but mastitis, a common disease during lactation, presents a major obstacle to fulfilling this recommendation. Maternal nutrient intake during lactation has been shown to be related to mastitis. Therefore, this study aimed to explore the effect of hesperetin, a phytonutrient, on mastitis. The oral administration of hesperetin to lipopolysaccharide (LPS)-induced mastitis mice alleviated their pathological damage, reduced the secretion of pro-inflammatory cytokines, and maintained the integrity of their blood-milk barrier. Moreover, our results showed that oral administration of hesperetin regulates the composition of the intestinal flora of mice. Fecal microbial transplantation (FMT) from the mice of hesperetin group alleviated LPS-induced mastitis in recipient mice. In additional, hesperetin attenuated the inflammatory response and increased the expression of tight junction proteins (TJs) in LPS-stimulated mouse mammary epithelial cells (mMECs). Through network pharmacological analysis and further research, we demonstrated hesperetin inhibits the expression of TLR4 and the activation of NF-κB signaling. In conclusion, hesperetin protects the blood-milk barrier and improve mastitis by regulating intestinal flora and inhibiting the activation of TLR4/NF-κB signaling axis. This study provides a theoretical basis for lactating females to consume hesperetin as a supplement to prevent mastitis and maintain mammary health.

The effect of bta-miR-1296 on the proliferation and extracellular matrix synthesis of bovine mammary fibroblasts.

Mammary fibrosis in dairy cows is a chronic condition caused by mastitis, and can lead to serious culling of dairy cows resulting in huge economic losses in the dairy industry. MicroRNAs (miRNAs) exert an important role in regulating mammary gland health in dairy cows. This study investigated whether exosomal miRNAs in mammary epithelial cells can regulate the proliferation of bovine mammary fibroblasts (BMFBs) in mastitis. Liposome transfection technology was used to construct a cellular model of the overexpression and inhibition of miRNAs. The STarMir software, dual luciferase reporter gene test, real-time quantitative PCR (qRT-PCR), a Cell Counting Kit-8 (CCK-8), and a Western Blot and plate clone formation test were used to investigate the mechanism by which bta-miR-1296 regulates the proliferation of BMFBs. Target gene prediction results revealed that glutamate-ammonia ligase was a direct target gene by which bta-miR-1296 regulates cell proliferation. It was found that bta-miR-1296 significantly inhibited the proliferation of BMFBs. After BMFBs were transfected with a bta-miR-1296 mimic, mRNA expression in the extracellular matrix (ECM), α-smooth muscle actin (α-SMA), collagen type I alpha 1 chain (COL1α1) and collagen type III alpha 1 chain (COL3α1), and various cell growth factors (basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-ß1 (TGF-ß1)) were down-regulated, and the expressions of α-SMA, COL1α1, COL3α1, phospho-extracellular regulated protein kinases, phospho-protein kinase B, TGF-ß1, and phospho-Smad family member3 proteins were inhibited. In conclusion, bta-miR-1296 can inhibit the proliferation of BMFBs and the synthesis of ECM in BMFBs, thus affecting the occurrence and development of mammary fibrosis in dairy cows and laying the foundation for further studies to clarify the regulatory mechanism of mammary fibrosis.

L-arginine attenuates Streptococcus uberis-induced inflammation by decreasing miR155 level.

L-arginine, as an essential substance of the immune system, plays a vital role in innate immunity. MiR155, a multi-functional microRNA, has gained importance as a regulator of homeostasis in immune cells. However, the immunoregulatory mechanism between L-arginine and miR155 in bacterial infections is unknown. Here, we investigated the potential role of miR155 in inflammation and the molecular regulatory mechanisms of L-arginine in Streptococcus uberis (S. uberis) infections. And we observed that miR155 was up-regulated after infection, accompanying the depletion of L-arginine, leading to metabolic disorders of amino acids and severe tissue damage. Mechanically, the upregulated miR155 mediated by the p65 protein played a pro-inflammatory role by suppressing the suppressor of cytokine signaling 6 (SOCS6)-mediated p65 ubiquitination and degradation. This culminated in a violently inflammatory response and tissue damage. Interestingly, a significant anti-inflammatory effect was revealed in L-arginine supplementation by reducing miR155 production via inhibiting p65. This work firstly uncovers the pro-inflammatory role of miR155 and an anti-inflammatory mechanism of L-arginine in S.uberis infection with a mouse mastitis model. Collectively, we provide new insights and strategies for the prevention and control of this important pathogen, which is of great significance for ensuring human food health and safety.

Novel lncRNA regulatory elements in milk somatic cells of Holstein dairy cows associated with mastitis.

Despite regulatory elements such as long non - coding RNAs representing most of the transcriptome, the functional understanding of long non - coding RNAs in relation to major health conditions including bovine mastitis is limited. This study examined the milk somatic cell transcriptome from udder quarters of 6 Holstein dairy cows to identify differentially expressed long non - coding RNAs using RNA - Sequencing. Ninety - four differentially expressed long non - coding RNAs are identified, 5 of which are previously annotated for gene name and length, 11 are annotated for gene name and 78 are novel, having no gene name or length previously annotated. Significant inflammatory response and regulation of immune response pathways (false discovery rate < 0.05) are associated with the differentially expressed long non - coding RNAs. QTL annotation analysis revealed 31 QTL previously annotated in the genomic regions of the 94 differentially expressed long non - coding RNAs, and the majority are associated with milk traits. This research provides a better understanding of long non - coding RNAs regulatory elements in milk somatic cells, which may enhance current breeding strategies for more adaptable or high mastitis resistant cattle.

Hexadecanamide alleviates Staphylococcus aureus-induced mastitis in mice by inhibiting inflammatory responses and restoring blood-milk barrier integrity.

Subacute ruminal acidosis (SARA) has been demonstrated to promote the development of mastitis, one of the most serious diseases in dairy farming worldwide, but the underlying mechanism is unclear. Using untargeted metabolomics, we found hexadecanamide (HEX) was significantly reduced in rumen fluid and milk from cows with SARA-associated mastitis. Herein, we aimed to assess the protective role of HEX in Staphylococcus aureus (S. aureus)- and SARA-induced mastitis and the underlying mechanism. We showed that HEX ameliorated S. aureus-induced mastitis in mice, which was related to the suppression of mammary inflammatory responses and repair of the blood-milk barrier. In vitro, HEX depressed S. aureus-induced activation of the NF-κB pathway and improved barrier integrity in mouse mammary epithelial cells (MMECs). In detail, HEX activated PPARα, which upregulated SIRT1 and subsequently inhibited NF-κB activation and inflammatory responses. In addition, ruminal microbiota transplantation from SARA cows (S-RMT) caused mastitis and aggravated S. aureus-induced mastitis, while these changes were reversed by HEX. Our findings indicate that HEX effectively attenuates S. aureus- and SARA-induced mastitis by limiting inflammation and repairing barrier integrity, ultimately highlighting the important role of host or microbiota metabolism in the pathogenesis of mastitis and providing a potential strategy for mastitis prevention.

ZEA mediates autophagy through the ROS-AMPK-m-TOR pathway to enhance the susceptibility of mastitis induced by Staphylococcus aureus in mice.

Mastitis is an inflammatory response of the mammary tissue caused by pathogenic bacterial infections, especially Staphylococcus aureus (S. aureus). Zearalenone (ZEA) is one of the common mycotoxins in moldy feed, which usually affects the cow's resistance to pathogenic microorganisms. However, it is not well understood whether ZEA affects the development of mastitis. Therefore, this study aimed to investigate the role of ZEA in the development of S. aureus-induced mastitis in mice. The results showed that administered daily by gavage for one week of ZEA (40 mg/kg) aggravated the severity of mastitis induced by S. aureus. Furthermore, we found that ZEA promotes the adhesion and invasion of S. aureus into mouse mammary epithelial cells (MMEC) by activating autophagy, and the activation of autophagy mediated by ROS-AMPK-m-TOR pathway. Taken together, the results showed that ZEA enhances S. aureus-induced mastitis susceptibility through activating autophagy mediated by ROS-AMPK-mTOR signaling pathway.

Acute phase reaction to lipopolysaccharide-induced mastitis in early lactation dairy cows fed nitrogenic, glucogenic, or lipogenic diets.

The availability of certain macronutrients is likely to influence the capacity of the immune system. Therefore, we investigated the acute phase response to intramammary (i.mam.) lipopolysaccharide (LPS) in dairy cows fed a nitrogenic diet (n = 10) high in crude protein, a glucogenic diet (n = 11) high in carbohydrates and glucogenic precursors, or a lipogenic diet (n = 11) high in lipids. Thirty-two dairy cows were fed one of the dietary concentrates directly after calving until the end of trial at 27 ± 3 days in milk (mean ± standard deviation). In wk 3 of lactation, 20 µg of LPS was i.mam. injected in one quarter, and sterile NaCl (0.9%) in the contralateral quarter. Milk samples of the LPS-challenged and control quarter were taken hourly from before (0 h) until 9 h after LPS challenge and analyzed for milk amyloid A (MAA), haptoglobin (HP), and IL-8. In addition, blood samples were taken in the morning, and composite milk samples at morning and evening milkings, from 1 d before until 3 d after LPS challenge, and again on d 9, to determine serum amyloid A (SAA) and HP in blood, and MAA and HP in milk. The mRNA abundance of various immunological and metabolic factors in blood leukocytes was quantified by quantitative reverse-transcription PCR from samples taken at -18, -1, 6, 9, and 23 h relative to LPS application. The dietary concentrates did not affect any of the parameters in blood, milk, and leukocytes. The IL-8 was increased from 2 h, HP from 2 to 3 h, and MAA from 6 h relative to the LPS administration in the milk of the challenged quarter and remained elevated until 9 h. The MAA and HP were also increased at 9 h after LPS challenge in whole-udder composite milk, whereas HP and SAA in blood were increased only after 23 h. All 4 parameters were decreased again on d 9. Similar for all groups, the mRNA abundance of HP and the heat shock protein family A increased after the LPS challenge, whereas the mRNA expression of the tumor necrosis factor α and the leukocyte integrin ß 2 subunit (CD18) were decreased at 6 h after LPS challenge. The glucose transporter (GLUT)1 mRNA abundance decreased after LPS, whereas that of the GLUT3 increased, and that of the GLUT4 was not detectable. The mRNA abundance of GAPDH was increased at 9 h after LPS and remained elevated. The acute phase protein response was detected earlier in milk compared with blood indicating mammary production. However, immunological responses to LPS were not affected by the availability of specific macronutrients provided by the different diets.

LC/MS-based untargeted lipidomics reveals lipid signatures of nonpuerperal mastitis.

BACKGROUND: Nonpuerperal mastitis (NPM) is a disease that presents with redness, swelling, heat, and pain during nonlactation and can often be confused with breast cancer. The etiology of NPM remains elusive; however, emerging clinical evidence suggests a potential involvement of lipid metabolism. METHOD: Liquid chromatography‒mass spectrometry (LC/MS)-based untargeted lipidomics analysis combined with multivariate statistics was performed to investigate the NPM lipid change in breast tissue. Twenty patients with NPM and 10 controls were enrolled in this study. RESULTS: The results revealed significant differences in lipidomics profiles, and a total of 16 subclasses with 14,012 different lipids were identified in positive and negative ion modes. Among these lipids, triglycerides (TGs), phosphatidylethanolamines (PEs) and cardiolipins (CLs) were the top three lipid components between the NPM and control groups. Subsequently, a total of 35 lipids were subjected to screening as potential biomarkers, and the chosen lipid biomarkers exhibited enhanced discriminatory capability between the two groups. Furthermore, pathway analysis elucidated that the aforementioned alterations in lipids were primarily associated with the arachidonic acid metabolic pathway. The correlation between distinct lipid populations and clinical phenotypes was assessed through weighted gene coexpression network analysis (WGCNA). CONCLUSIONS: This study demonstrates that untargeted lipidomics assays conducted on breast tissue samples from patients with NPM exhibit noteworthy alterations in lipidomes. The findings of this study highlight the substantial involvement of arachidonic acid metabolism in lipid metabolism within the context of NPM. Consequently, this study offers valuable insights that can contribute to a more comprehensive comprehension of NPM in subsequent investigations. TRIAL REGISTRATION: Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Number: 2019-702-57; Date: July 2019).

Association of Nonpuerperal Mastitis with Cytokines Related to Helper T Cells TH1/TH2 and TH17/Treg.

Objective: To investigate the association of nonpuerperal mastitis with cytokines related to the helper T cells TH1/TH2 and TH17/Treg and associated immune balance. Methods: From 2016 to 2021, we included 40 patients with non-puerperal mastitis who underwent surgery at China-Japan Friendship Hospital and compared them with 40 control patients with benign non-infectious breast disease. Hematoxylin-eosin staining detects inflammatory infiltrates of breast tissue. The expression of interferon γ and interleukin 4 in breast tissue was detected by immunofluorescence imaging, and the relative protein expression of TH1/TH2 and TH17/Treg cell-associated cytokines in CD4+ T cells was detected by western blotting. CD4+ T cells were isolated by fluorescence-activated cell sorting for detection of the relative protein expression of interferon γ and interleukin 4 in CD4+ T cells. Results: Hematoxylin-eosin staining showed that the nonpuerperal mastitis group had significantly greater inflammatory infiltration than the control group. Immunofluorescence images showed the relative fluorescence intensity of interferon γ was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative fluorescence intensity of interleukin 4 did not significantly differ between the 2 groups (P = .0686). Western blotting revealed that the relative protein expression of interferon γ, interleukin 2, and interleukin 17 was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative protein expression of interleukin 4 (P = .0512), interleukin 10 (P = .3088), and transforming growth factor ß (P = .0653) did not significantly differ between the 2 groups. Flow cytometry of isolated CD4+ T cells showed the relative protein expression of interferon γ was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative protein expression of interleukin 4 did not significantly differ between the 2 groups (P = .0680). Conclusion: The expression of the TH1 cytokines interferon γ and interleukin 2 and the TH17 cytokine interleukin 17 was significantly higher in patients with nonpuerperal mastitis, while the TH2 cytokine interleukin 4 and the Treg cytokines interleukin 10 and transforming growth factor ß were expressed at lower levels. This study provides new research ideas for the treatment of mastitis.

Saikosaponin A alleviates Staphylococcus aureus-induced mastitis in mice by inhibiting ferroptosis via SIRT1/Nrf2 pathway.

Mastitis is a common and serious bacterial infection of the mammary gland. Saikosaponin A (SSA) is a triterpenoid saponin isolated from Bupleurum falcatum that has the ability to treat various diseases. However, little is known about the role of SSA in achieving mastitis remission. Here, we found that SSA alleviated Staphylococcus aureus (S. aureus)-induced mastitis by attenuating inflammation and maintaining blood-milk barrier integrity. Furthermore, S. aureus activated nuclear factor kappa B (NF-κB) pathway by upregulated p-p65 and p-IκB. S. aureus also induced ferroptosis in mammary gland in mice, mainly characterized by excessive iron accumulation, mitochondrial morphological changes and impaired antioxidant production. However, S. aureus-induced NF-κB activation and ferroptosis were prevented by SSA. Moreover, SAA could upregulate the expression of SIRT1, Nrf2, HO-1 and GPX4. And the inhibitory effects of SAA on inflammation and ferroptosis were reversed by SIRT1 inhibitor EX-527. In conclusion, SAA protected S. aureus-induced mastitis through suppressing inflammation and ferroptosis by activating SIRT1/Nrf2 pathway.

Relationships between body- and udder-related type traits with somatic cell counts and potential use for an early selection method for water buffaloes (Bubalus bubalis).

Water buffalo milk is a reliable source of high-quality nutrients; however, the susceptibility of mastitis in buffaloes must be taken into consideration. An animal with somatic cell count (SCC) of greater than 250,000 cells/mL is reported to be likely to have mastitis which has serious adverse effects on animal health, reproduction, milk yield, and milk quality. Type traits (TTs) of water buffalo can affect SCC in animal milk to some extent, but few reports on the correlation between SCC and TTs are available. In this study, a total of 1908 records collected from 678 water buffaloes were investigated. The general linear model was used to identify factors associated with phenotypic variation of the somatic cell score (SCS) trait, including parity, lactation length, calving year, and calving season as fixed effects. Using PROC CORR analysis method, taking calving year and lactation length as covariates, the correlation co-efficient between TT and SCS was obtained. Our results showed that correlation co-efficients between the 45 TTs with SCS ranged from 0.003 to 0.443 (degree of correlation). The correlation between udder traits and SCS was greater than that between body structure traits and SCS. Among udder traits, distance between teats (including front and rear teat distance [r = 0.308], front teat distance [r = 0.211], and teat crossing distance [r = 0.412]) and teat circumference (r = 0.443) had the highest correlation with SCS, followed by the leg traits including rear leg height (r = -0.354) and hock bend angle (r = -0.170). Animal with high rear legs (>48 cm) and short teat crossing distance (<17 cm), and narrow teat circumference (<11 cm) exhibited low SCS. Using four nonlinear models (Von Bertalanffy, Brody, Logistic, and Gompertz), the optimal growth curves of the TTs highly correlated with the SCS (rear leg height and teat crossing distance) were fitted, and the correction co-efficients of these two TTs rear leg height and teat crossing distance of animal from young age (2 mo old) to first lactation (35 mo old) were attained for establishment of early selection method for water buffaloes with low SCS. This study provides theoretical support for early selection of low-SCS water buffaloes and lays a foundation for improving milk quality and promoting healthy development of water buffalo's dairy industry.

Some type traits (TTs) have been reported to affect somatic cell count (SCC) in bovine milk, which result in mastitis to some extent, but the correlation between SCC and TTs of water buffalo has been poorly understood. Here, a total of 1908 records from 678 buffaloes were investigated. The correlation between 45 TTs and somatic cell score (SCS) was analysed, and the optimal growth curves of TTs highly correlated with SCS were fitted. Our result showed that high rear legs (>48 cm) and short teat crossing distance (<17 cm), and narrow teat circumference (<11 cm) were correlated with low SCS. We obtained correction co-efficients for two TTs highly correlated with SCS of water buffalos from young age (2 mo old) to first lactation (35 mo) by fitting the optimal growth curve for rear leg height and teat crossing distance. This study provides theoretical support for early selection for water buffaloes that are less susceptible to mastitis, and lays a foundation for improving milk quality and promoting healthy development of water buffalo dairy industry.

Maslinic acid alleviates LPS-induced mice mastitis by inhibiting inflammatory response, maintaining the integrity of the blood-milk barrier and regulating intestinal flora.

Mastitis occurs frequently in breastfeeding women and not only affects the women's health but also hinders breastfeeding. Maslinic acid is a type of pentacyclic triterpenoid widely found in olives that has good anti-inflammatory activity. This study aims to discuss the protective function of maslinic acid against mastitis and its underlying mechanism. For this, mice models of mastitis were established using lipopolysaccharide (LPS). The results revealed that maslinic acid reduced the pathological lesions in the mammary gland. In addition, it reduced the generation of pro-inflammatory factors and enzymes (IL-6, IL-1ß, TNF-α, iNOS, and COX2) in both mice mammary tissue and mammary epithelial cells. The high-throughput 16S rDNA sequencing of intestinal flora showed that in mice with mastitis, maslinic acid treatment altered ß-diversity and regulated microbial structure by increasing the abundance of probiotics such as Enterobacteriaceae and downregulating harmful bacteria such as Streptococcaceae. In addition, maslinic acid protected the blood-milk barrier by maintaining tight-junction protein expression. Furthermore, maslinic acid downregulated mammary inflammation by inhibiting the activation of NLRP3 inflammasome, AKT/NF-κB, and MAPK signaling pathways. Thus, in a mice model of LPS-induced mastitis, maslinic acid can inhibit the inflammatory response, protect the blood-milk barrier, and regulate the constitution of intestinal flora.

Unique Peptides of Cathelicidin-1 in the Early Detection of Mastitis-In Silico Analysis.

Based on the results of previously performed clinical studies, cathelicidin-1 has been proposed as a potential biomarker for the early diagnosis of mastitis in ewes. It has been hypothesized that the detection of unique peptides (defined as a peptide, irrespective of its length, that exists in only one protein of a proteome of interest) and core unique peptides (CUPs) (representing the shortest peptide that is unique) of cathelicidin-1 may potentially improve its identification and consequently the diagnosis of sheep mastitis. Peptides of sizes larger than those of the size of CUPs, which include consecutive or over-lapping CUPs, have been defined as 'composite core unique peptides' (CCUPs). The primary objective of the present study was the investigation of the sequence of cathelicidin-1 detected in ewes' milk in order to identify its unique peptides and core unique peptides, which would reveal potential targets for accurate detection of the protein. An additional objective was the detection of unique sequences among the tryptic digest peptides of cathelicidin-1, which would improve accuracy of identification of the protein when performing targeted MS-based proteomics. The potential uniqueness of each peptide of cathelicidin-1 was investigated using a bioinformatics tool built on a big data algorithm. A set of CUPs was created and CCUPs were also searched. Further, the unique sequences in the tryptic digest peptides of cathelicidin-1 were also detected. Finally, the 3D structure of the protein was analyzed from predicted models of proteins. In total, 59 CUPs and four CCUPs were detected in cathelicidin-1 of sheep origin. Among tryptic digest peptides, there were six peptides that were unique in that protein. After 3D structure analysis of the protein, 35 CUPs were found on the core of cathelicidin-1 of sheep origin and among them, 29 were located on amino acids in regions of the protein with 'very high' or 'confident' estimates of confidence of the structure. Ultimately, the following six CUPs: QLNEQ, NEQS, EQSSE, QSSEP, EDPD, DPDS, are proposed as potential antigenic targets for cathelicidin-1 of sheep. Moreover, another six unique peptides were detected in tryptic digests and offer novel mass tags to facilitate the detection of cathelicidin-1 during MS-based diagnostics.

Resveratrol inhibits LPS-induced apoptosis in bovine mammary epithelial cells: the role of PGC1α-SIRT3 axis.

Resveratrol (Res) is a bioactive dietary component and alleviates apoptosis in multiple cell types. However, its effect and mechanism on lipopolysaccharide (LPS)-induced bovine mammary epithelial cells (BMEC) apoptosis, which commonly happens in dairy cows with mastitis, is unknown. We hypothesized that Res would inhibit LPS-induced apoptosis in BMEC through SIRT3, a NAD + -dependent deacetylase activated by Res. To test the dose-response effect on apoptosis, 0-50 µM Res were incubated with BMEC for 12 h, followed by 250 µg/mL LPS treatment for 12 h. To investigate the role of SIRT3 in Res-mediated alleviation of apoptosis, BMEC were pretreated with 50 µM Res for 12 h, then incubated with si-SIRT3 for 12 h and were finally treated with 250 µg/mL LPS for 12 h. Res dose-dependently promoted the cell viability and protein levels of Bcl-2 (Linear P < 0.001) but decreased protein levels of Bax, Caspase-3 and Bax/Bcl-2 (Linear P < 0.001). TUNEL assays indicated that cellular fluorescence intensity declined with the rising doses of Res. Res also dose-dependently upregulated SIRT3 expression, but LPS had the opposite effect. SIRT3 silencing abolished these results with Res incubation. Mechanically, Res enhanced the nuclear translocation of PGC1α, the transcriptional cofactor for SIRT3. Further molecular docking analysis revealed that Res could directly bind to PGC1α by forming a hydrogen bond with Tyr-722. Overall, our data suggested that Res relieved LPS-induced BMEC apoptosis through the PGC1α-SIRT3 axis, providing a basis for further in vivo investigations of applying Res to relieve mastitis in dairy cows.

Puerarin Alleviates H2O2-Induced Oxidative Stress and Blood-Milk Barrier Impairment in Dairy Cows.

During the perinatal period, the bovine mammary epithelial cells of dairy cows exhibit vigorous metabolism and produce large amounts of reactive oxygen species (ROS). The resulting redox balance disruption leads to oxidative stress, one of the main causes of mastitis. Puerarin (PUE) is a natural flavonoid in the root of PUE that has attracted extensive attention as a potential antioxidant. This study first investigated whether PUE could reduce oxidative damage and mastitis induced by hydrogen peroxide (H2O2) in bovine mammary epithelial cells in vitro and elucidated the molecular mechanism. In vitro, BMECs (Bovine mammary epithelial cells) were divided into four treatment groups: Control group (no treatment), H2O2 group (H2O2 stimulation), PUE + H2O2 group (H2O2 stimulation before PUE rescue) and PUE group (positive control). The growth of BMECs in each group was observed, and oxidative stress-related indices were detected. Fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of tightly linked genes, antioxidant genes, and inflammatory factors. The expression of p65 protein was detected by Western blot. In vivo, twenty cows with an average age of 5 years having given birth three times were divided into the normal dairy cow group, normal dairy cow group fed PUE, mastitis dairy cow group fed PUE, and mastitis dairy cow group fed PUE (n = 5). The contents of TNF-α, IL-6, and IL-1ß in milk and serum were detected. In BMECs, the results showed that the PUE treatment increased the activities of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC); ROS and malondialdehyde (MDA) levels were reduced. Thus, PUE alleviated H2O2-induced oxidative stress in vitro. In addition, the PUE treatment eliminated the inhibition of H2O2 on the expression of oxidation genes and tight junction genes, and the enrichment degree of NRF-2, HO-1, xCT, and tight junctions (claudin4, occludin, ZO-1 and symplekin) increased. The PUE treatment also inhibited the expression of NF-κB-associated inflammatory factors (IL-6 and IL-8) and the chemokine CCL5 in H2O2-induced BMECs. In vivo experiments also confirmed that feeding PUE can reduce the expression of inflammatory factors in the milk and serum of lactating dairy cows. In conclusion, PUE can effectively reduce the oxidative stress of bovine mammary epithelial cells, enhance the tight junctions between cells, and play an anti-inflammatory role. This study provides a theoretical basis for PUE prevention and treatment of mastitis and oxidative stress. The use of PUE should be considered as a feed additive in future dairy farming.

An herbal drug combination identified by knowledge graph alleviates the clinical symptoms of plasma cell mastitis patients: A nonrandomized controlled trial.

Background: Plasma cell mastitis (PCM) is a nonbacterial breast inflammation with severe and intense clinical manifestation, yet treatment methods for PCM are still rather limited. Although the mechanism of PCM remains unclear, mounting evidence suggests that the dysregulation of immune system is closely associated with the pathogenesis of PCM. Drug combinations or combination therapy could exert improved efficacy and reduced toxicity by hitting multiple discrete cellular targets. Methods: We have developed a knowledge graph architecture toward immunotherapy and systematic immunity that consists of herbal drug-target interactions with a novel scoring system to select drug combinations based on target-hitting rates and phenotype relativeness. To this end, we employed this knowledge graph to identify an herbal drug combination for PCM and we subsequently evaluated the efficacy of the herbal drug combination in clinical trial. Results: Our clinical data suggests that the herbal drug combination could significantly reduce the serum level of various inflammatory cytokines, downregulate serum IgA and IgG level, reduce the recurrence rate, and reverse the clinical symptoms of PCM patients with improvements in general health status. Conclusions: In summary, we reported that an herbal drug combination identified by knowledge graph can alleviate the clinical symptoms of PCM patients. We demonstrated that the herbal drug combination holds great promise as an effective remedy for PCM, acting through the regulation of immunoinflammatory pathways and improvement of systematic immune level. In particular, the herbal drug combination could significantly reduce the recurrence rate of PCM, a major obstacle to PCM treatment. Our data suggests that the herbal drug combination is expected to feature prominently in future PCM treatment. Funding: C. Liu's lab was supported by grants from the Public Health Science and Technology Project of Shenyang (grant: 22-321-32-18); Y. Yang's laboratory was supported by the National Natural Science Foundation of China (grant: 81874301), the Fundamental Research Funds for Central University (grant: DUT22YG122), and the Key Research project of 'be Recruited and be in Command' in Liaoning Province (2021JH1/10400050). Clinical trial number: NCT05530226.