Effect of anemoside B4 on milk whey in clinical mastitis-affected cows elucidated using tandem mass tag (TMT)-based quantitative proteomics

doi:10.21203/rs.3.rs-1504389/v1

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Effect of anemoside B4 on milk whey in clinical mastitis-affected cows elucidated using tandem mass tag (TMT)-based quantitative proteomics

https://doi.org/10.21203/rs.3.rs-1504389/v1

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Objective

Intramuscular injection of anemoside B4 (AB4) has a superior therapeutic effect on clinical mastitis in lactating cows. Here, we explored AB4’s effect on milk whey in clinical mastitis-affected cows using proteomics.

Methods

Among fifty clinical mastitis cows received AB4 administration (0.05ml/kg/day, for seven days), twelve healed cows were selected and marked as group T. Twelve clinically heathy cows received the same dose of saline for seven days, marked as group C. Collected milk whey of group T before and after AB4 administration marked as T1 and T2, respectively. And the milk whey of group C after saline injection marked as C1. Milk whey protein changes were detected with tandem mass tag-based quantitative proteomic.

Results

We identified 872 quantifiable proteins in the samples. Among them, 511 proteins between T1 and C1, and 361 proteins between T2 and T1 were significantly altered. T1 than C1 had significantly more proteins associated with inflammatory damage and trans-endothelial migration of leukocytes, whereas these proteins were reduced in T2 treated with AB4. Compared with C, proteins associated with fibrin clot degradation and complement system activation were downregulated in T1 but upregulated in T2.

Conclusion

In summary, AB4 can exert its therapeutic effect on clinical mastitis in cows mainly by reducing inflammatory damage, activating the complement system, inhibiting trans-endothelial migration of leukocytes, and promoting degradation of milk fibrin clots.

anemoside B4

milk whey

clinical mastitis

proteomics

Pulsatilla chinensis (Bunge) Regel, (a synonym of Anemone chinensis Bunge) belongs to the family of Ranunculaceae, being used as a kind of medical herb for a long history. In traditional Chinese medicine (TCM), P. chinensis is used to clear heat in body and detoxify, to cool blood and stop dysentery such as bacterial dysentery, amebic dysentery, especially good at clearing damp-heat of gastrointestinal tract and blood-heat toxin, in addition, it was reported to treat malaria and relieve spasm and pain as well¹. Modern pharmacological studies confirmed that the extracts or the active compounds, such as anemoside B4 (AB4), anemoside B5 (AB5), isolated from P. chinensis played important roles in anti-inflammatory, anti-microbial and antiviral^2,3. AB4 can inhibit the increase of TNF-α and E-selectin secretion from RIMECs caused by LPS with IL-6 unchanged, inhibits both LPS and concanavalin A-induced splenic lymphocyte proliferation, and reduces LPS-induced NF-κB activation and IL-6 production^4,5. The in vivo anti-inflammatory activities of AB4 assessed through the intraperitoneal injection of 2 mg/kg AB4 in mice showed protection against LPS-induced acute lung injury, accompanied by reduction of IL-6, neutrophil infiltration, and NF-κB activation⁶. AB4 (12.5–50 mg/kg) significantly suppresses xylene-induced mouse ear edema and ameliorated LPS-induced kidney and lung inflammation damage, inhibiting pro-inflammatory response by NF-κB pathway in mice⁴. Findings from these in vitro and in vivo studies demonstrate the anti-inflammatory and immunomodulatory effects of AB4, highlighting its utility as a potential therapy for inflammation and immune response-related conditions, which is clearly related to its’ traditional use.

Mastitis, with an incidence and prevalence ranging from 20% to 60 in dairy herds, respectively is the most common disease in dairy cows⁷. It results in considerable monetary losses to the dairy industry due to decreased milk yield and quality, reduced reproductive performance, treatment, and premature culling^7–9. Mastitis is the inflammation of the mammary gland, typically arising due to intramammary infections, broadly classified into subclinical (SCM) and clinical mastitis (CM) based on the severity of infection¹⁰. CM displays symptoms such as fever, depression, and anorexia and is usually associated with visible local and systemic signs of inflammation, marked by changes in milk, such as the appearance of clots, flakes, or watery texture^11,12. CM is mainly caused by infection with pathogenic microorganisms such as Escherichia coli, Streptococcus agalactiae, and Staphylococcus aureus. E. coli releases bacterial cell wall components, such as lipopolysaccharide (LPS), which induces markedly increased local inflammatory mediators with a strong systemic acute phase response¹³. Activation of TLR4-dependent signaling pathways by LPS results in either the production of proinflammatory factors or initiation of endothelial apoptosis. Blocking TLR4 activation may inhibit the inflammatory response caused by LPS¹⁴. The S. agalactiae infection may affect cell proliferation and milk composition by reducing immune cell migration and inhibiting ATP synthesis and pathogen recognition pathways, thereby preventing immune response⁷. S. aureus is a gram-positive, opportunistic, and contagious bacterium that is one of the most prevalent causes of mastitis in cattle¹⁵. S. aureus-induced mastitis is difficult to eradicate with available therapies and is prone to persistent or recurrent infections due to the mechanisms that escape the attack from the host’s immune¹⁶. The desire to eradicate pathogens and assist the immune response established the routine use of intramammary antibiotics for the treatment of CM¹⁷. However, the extensive use of antibiotics has led to the evolution of antibiotic resistance in many bacterial species¹⁸. Therefore, government regulations have encouraged reduced antibiotic usage.

Previous studies indicate that intramuscular AB4 injection significantly attenuates clinical signs and manifestations in dairy cows with mastitis(Qian et al., 2021). Here, we analyzed milk whey from dairy cows intramuscularly injected with AB4 after natural infection of CM using a tandem mass tag (TMT) proteomic approach and aimed to detect target proteins and molecular pathways associated with the therapeutic effect of AB4.

ARRIVE statement

This study was carried out in compliance with the ARRIVE guidelines.

Ethics approval and consent to participate

All experiments were performed in accordance with Ethics Statement Approval of Institute Animal Ethics (IAEC) committee guidelines and reviewed by the Institutional Animal Care and Use Committee of Sichuan Agricultural University (DYY-S20174610).

Animal and treatment

Fifty natural infected CM Chinese Holstein cows, identified and confirmed by a veterinary surgeon according to the criteria of Turk¹¹, with SCC above 500,000 cells/mL, positive CMT, clinical signs of mastitis, which included changes in milk appearance (flakes and clots in milk), different stages of udder inflammation (hyperemia, edema, pain, udder enlargement, and elevated udder temperature), and a disturbance of general health (depression, relaxed cold ears, dehydration, elevated body temperature, increased heart and respiratory rates, and decreased ruminal contraction and appetite), were recruited to the study from a dairy farm in the region of Southern Sichuan Province. All CM cows received an intramuscular infusion of AB4 (Guangxi innovates medical technology Co., Ltd. Lipu, Guangxi, China) (0.05 mL/kg, once daily) for seven days. During this period, clinical symptoms and milk traits were observed every day to judge the therapeutic effect. CM cows without clinical symptoms, such as change in the appearance of milk (color, viscosity, consistency; i.e., flaky sediments, watery appearance, discoloration) and local clinical signs of inflammation of the udder parenchyma (i.e., swelling, heat, pain, redness), were assessed as clinically cured. Among the clinical cured cows, twelve clinical cured cows, at the 2^nd-3^rd lactation stage, weighted 565-659 kg and aged 3-4 years, were selected and marked as group T. Before (T1) and after (T2) AB4 injection, 10 mL milk samples were collected from groups T. Twelve health cows with same lactation stage, body weight and age, received an intramuscular injection of same dose saline for seven days, marked as group C. Collected 10 mL milk samples of group C after saline injection (C1). All of these cows were fed a diet composed of 60% corn silage and hay 40% concentrate containing crude protein, raw fat, and minerals.

Sample collection and milk whey preparation

Scrub teats with a pad soaked in 70% alcohol (Chengdu Kolon Chemicals Co., CN) and dried; massage the udder and discard the first three streams of milk from the teat before sampling. Collected milk samples was centrifuged at 10 000 × g for 10 min to remove the cream layer (MTX 150 centrifuge, Thermo Fisher Scientific, USA). Then, CaCl₂ (Chengdu Kolon Chemicals Co., CN) was added to a final concentration of 60 mM. The skim milk was acidified at pH 4.6 with the addition of 10% hydrochloric acid (Chengdu Kolon Chemicals Co., CN) to precipitate casein. The whey protein was centrifuged at 189 000 × g for 60 min. The supernatant was collected for further TMT analysis. Three sample pools of each group were prepared by mixing milk whey from the four dairy cows. Meanwhile, SCC and inflammatory factors (IL-1β, IL-6, IL-8, IL-10, TNF-α) in whey were detected by SCC counter (Zhong Lao Technologies Ltd., CN) and Elisa kit (Nanjing Jiancheng Bioengineering Institution., CN) to evaluate the effectiveness of AB4 treatment.

Protein digestion and TMT labeling

Sample proteins were extracted using the SDT method (4% w/v SDS, 100 mM Tris/HCl pH 7.6, 0.1M DTT)²⁰. Protein concentration was determined using the BCA kit (Thermo Fisher Scientific, USA). Proteins were digested using filter-aided proteome preparation (FASP) with trypsin (Solarbio, CN) hydrolysis²⁰. The peptides derived from pooled samples were labeled using a TMT kit (Thermo Fisher Scientific, USA).

Fractionation of labeled peptides using high pH reversed-phase column

A C18 column (Thermo Scientific Acclaim PepMap100, 100 μm ´ 2cm, nanoViper C18, USA) was preequilibrated with 0.1% (v/v) trifluoroacetic acid (Solarbio, CN) and eluted using an acetonitrile (containing 0.1% trifluoroacetic acid) gradient. The labeled peptides were loaded onto the column, followed by the addition of miller water. The mixture was centrifuged at a low speed to desalt (Micro 17, Thermo Fisher Scientific, USA). Elute the peptide from the C18 column using a gradient of acetonitrile. Digested peptide samples were dried in vacuum freeze dryer (LyoQest-55, Telstar, Spain) and reconstituted in fluoroacetic acid (Chengdu Kolon Chemicals Co., CN). The peptide concentration was subsequently detected at OD280 (Nano Drop One^C, Thermo Fisher Scientific, USA).

High-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis

LC-MS/MS analysis of TMT-labeled peptides was carried out using an HPLC system (Thermo Fisher Scientific, USA) coupled to an Easy nLC system (Thermo Fisher Scientific, USA). TMT-labeled peptides were dissolved in loading solvent (1% acetonitrile, 16%; 0.1% formic acid, 84%) (Chengdu Kolon Chemicals Co., CN), loaded onto a C18 column, and separated on an analytical column (Thermo Fisher Scientific EASY column, 10 cm, ID, 75 μm, 3 μm, C18-A2, USA) at a flow rate of 300 μL/min. The MS was operated in positive ion mode using Q-Exactive. Full-scan MS spectra were acquired in the range from 300-1800 m/z with a resolution of 200-70 000 m/z, automated gain control (AGC) target 1e6, maximum IT 50 ms, and dynamic exclusion 60 sec. The 20 strongest signals of the parent ions were selected for secondary fragmentation. The secondary mass spectrometry resolution was 35 000 at 100 m/z (TMT 10-plex), and the MS2 activation type was high-energy collision dissociation (HCD). The isolation window was 2 m/z and the normalized collision energy was 30 eV. Mascot 2.2 and Proteome Discoverer 1.4 were used to analyze the RAW data of the mass spectrometry results. The relevant parameters and descriptions are as follows: enzyme, trypsin; max missed cleavages, 2; fixed modifications, Carbamidomethyl (C), TMT 6/10plex (N-term),TMT6/10 plex (K); peptide mass tolerance, ± 20 ppm; fragment mass tolerance, 0.1Da; peptide FDR, ≤0.01. Proteins’ GenInfo identifier (GI) accession numbers were converted into official gene symbol by uniport (https://www.uniprot.org/)-Bos-taurus-20191014.

Data and statistical analysis

Gene ontology (GO database, http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG database, http://www.genome.jp/kegg/) pathway analysis was performed using Blast2Go (https://www.blast2go.com/). Fisher’s exact test was used to analyze the GO and KEGG functional enrichments. Functional network analysis was performed using STRING (http://string-db.org/) and Cytoscape platform version 3.7.2 (https://cytoscape.org) based on bos-taurus genes.

Clinical cure rate of AB4 in CM

The overall clinical cure rate of AB4 in CM was 76% (38/50).

Milk whey protein changes

We identified 1677 quantifiable proteins using a TMT-based quantitative proteomics approach according to the set criteria (two unique peptides and ≤1% FDR). Supplemental Table S1 provides a complete list of the identified proteins. The differentially expressed proteins (DEPs) between each group were defined based on a 1.2-fold change threshold (with a fold change >1.2 and <0.83, P<0.05) according to mass spectrum data (Figure 1). Supplemental Table S2 provided a full list of the DEPs. A total of 511 DEPs (268 upregulated and 263 downregulated) were detected between T1 and C1 (Figure 1A). The results showed that 361 DEPs were identified between T2 and T1, 177 proteins were upregulated, and 184 proteins were downregulated (Figure 1B).

GO analyses

We performed statistics on the distribution of DEPs using GO secondary annotation classification, including three major classes: biological processes (BP), cellular components (CC), and molecular functions. Among the DEPs of T1/C1, BP included biological regulation, stress response, organonitrogen compound metabolic process, etc. MF included nucleosomal DNA binding, chromatin DNA binding, and protein binding. CC included the Golgi membrane, nuclear chromosome, chromatin, etc. (Supplemental Table S3). Among the DEPs of T2/T1, BP included a response to lipid, interleukin-1 beta production, regulation of interleukin-1 production, etc. MF included serine-type endopeptidase activity, cytoskeletal protein binding, and transforming growth factor beta receptor binding. CC included the extracellular region, cytoplasm, and the cortical cytoskeleton (Supplemental Table S4). The top 20 enriched GO terms of T1/C1 and T2/T1 are shown in Figure 2.

KEGG analyses

KEGG enrichment analysis results are shown in Supplemental Table S5. DEPs of T1/C1 enriched in 21 pathways and the top 20 are shown in Figure 3B: complement and coagulation cascades, pyruvate metabolism, leukocyte transendothelial migration, regulation of actin cytoskeleton, etc. Additionally, DEPs of T2/T1 were enriched in 25 pathways (Figure 3) andthetop 20 pathways included focal adhesion, leukocyte transendothelial migration, regulation of actin cytoskeleton, etc. There were 10 common enriched pathways of T1/C1 and T2/T1: pyruvate metabolism, Wnt signaling pathway, pentose phosphate pathway, glycolysis/gluconeogenesis, HIF-1 signaling pathway, central carbon metabolism in cancer, leukocyte transendothelial migration, regulation of actin cytoskeleton, Fc gamma R-mediated phagocytosis, and viral carcinogenesis. Besides complement and coagulation cascades had 18 proteins in T1/C1 and eight proteins in T2/T1. In summary, the above mentioned 11 pathways were considered to have a potential role in AB4 (Table 1).

Target proteins analysis

Among the 268 upregulated proteins in dairy cows with CM (T1/C1), 164 were downregulated after AB4 treatment (Figure 4A). Additionally, 243 proteins were downregulated in cows with CM, 114 of which were upregulated following AB4 administration (Figure 4B). These 274 proteins were potential targets for AB4. The PPI network revealed potential connections between the targets. Following the removal of free proteins, the PPI network contained 167 nodes and 5116 edges, with an average node degree of 10.07. The color of a node reflected the degree of importance. The larger the degree, the more important the node was in the network, suggesting that it may be a key target of B4 treatment. According to the degree value, hub proteins were defined as proteins with a degree greater than 20, as shown in Figure 5 (red nodes).

We previously demonstrated that an IM injection of AB4 was therapeutically effective in naturally infected clinic mastitis in dairy cows(Qian et al., 2021). Here, we identified 872 proteins in milk whey from dairy cows using the TMT proteomic approach. Among these proteins, 361 proteins significantly changed after IM injection of B4 in dairy cows with CM. These changes in proteins might be caused by the anti-inflammatory effect of AB4 on mastitis in dairy cows.

CM is caused by the invasion of pathogens. Gram-negative bacteria, such as E. coli, invade the mammary gland with LPS release, and the TLR4/nuclear factor-κB (NF-κB) signaling pathway is activated to produce pro-inflammatory factors and APPs, and cause mastitis²¹. S. aureus colonizes the mammary gland, adheres to the host epithelial cells and their extracellular matrix, synthesizes and secretes factors that allow the invasion, penetration, and destruction of the mammary tissue, including several exotoxins (hemolysins and leukocidins) and various hydrolytic enzymes such as proteases, coagulase, lipases, and hyaluronidases^22,23. Additionally, S. aureus can escape, and also modulate the host immune system by producing a range of factors such as S. aureus superantigen toxins, protein A, and polysaccharide capsule²⁴.

Romana et al. (2021) found that proteins involved in host defense such as α2- macroglobulin (A2M), α1-microglobulin/bikunin protein (AMBP), proteins involved in transport such as apolipoproteins AII and F (APOA2, APOF), and retinol-binding protein 4 (RBP4), are increased in milk but reduced in the serum of mastitis cows²⁵. However, we found that APOA1, APOA4, APOE, and RBP4 were downregulated during clinical mastitis. These proteins were transferred from the blood to the pathogens causing mastitis. When the consumption of these proteins was greater than the rate of transfer from the blood, this may have caused conflicting results. This indicated that changes in the abundance levels of these proteins were not sensitive enough to act as biomarkers to predict infection of the mammary gland. Proteins involved in innate immunity and antimicrobial functions (e.g., serotransferrin, complement C3, fibrinogen gamma-B chain, and cathepsin B) and are associated with the immune response to pathogens (e.g., polymeric immunoglobulin receptor-like protein, MHC class I antigen, and beta-2-microglobulin) are abundantly expressed in whey from S. aureus mastitis milk²⁶. HP, SAA1, DEFB10, and SERPINB3 are also observed to increase in milk from cows with mastitis²⁷. Sudipa et al. Changes in the expression of HP and FN from Holstein Friesian correlated with disease progression, and angiogenin and cofilin-1 were upregulated while ubiquitin family members were downregulated during disease transition²⁸. Proteins that stand out as logical candidates for further analyses include various APPs and vascular-derived proteins such as C3, C4, TF, ALB, TTR, FGA, and ITIH4 in mastitis whey of E. coli infected cows by 2-DE and label-free methods, respectively^29,30. Host defense-related proteins such as HP, SERPINB1, SERPINB4, and ITIH4 were upregulated in our study, consistent with previous studies.

DEPs of S. agalactiae-induced mastitis are mostly enriched in complement and coagulation cascades, glycolysis/gluconeogenesis, pentose phosphate pathway, purine metabolism, NOD-like receptor signaling pathway, inflammatory mediator regulation of TRP channels, bacterial invasion of epithelial cells, chemokine signaling pathway proteasome, cell adhesion molecules (CAMs), and ribosomes^7,31. S. aureus-infected mastitis also detected complement and coagulation cascades, pentose phosphate pathway, Fc gamma R-mediated phagocytosis pathway, leukocyte transendothelial migration pathway, acute phase response signaling, LXR/RXR activation, antigen processing and presentation pathway, and ECM–receptor interaction pathway ^26,32,33. E. coli-induced mastitis also found complement and coagulation cascades pathway, glycolysis/gluconeogenesis, pentose phosphate pathway, acute phase response signaling, lysosome, cytokine-cytokine receptor interaction, and cell adhesion molecules.

The top canonical pathways detected in E.coli were complement and coagulation cascades, glycolysis/gluconeogenesis, pentose phosphate pathway, leukocyte transendothelial migration^32,34. Most of the AB4-targeted pathways identified here were consistent with the above pathways, including glycolysis/gluconeogenesis, pentose phosphate pathway, leukocyte transendothelial migration pathway, Fc gamma R-mediated phagocytosis, regulation of actin cytoskeleton, and complement and coagulation cascades.

Glycolysis is the process of converting glucose into pyruvate and generating small amounts of ATP (energy) and NADH (reducing power)³⁵. Gluconeogenesis is a synthesis pathway of glucose from no carbohydrate precursors, and it is essentially a reversal of glycolysis with minor variations in alternative paths³⁶. When mastitis developed (T1/C1), GPI, TPI1, GAPDH, PGK1, PGAM1, ENO1, and PKM in the glycolysis/gluconeogenesis pathway were upregulated, which promoted pyruvate synthesis with large amounts of ATP production. ATP is present in inflamed tissues in vivo at extracellular concentrations sufficient for P2 receptor activation, promoting leukocyte recruitment and NALP3-inflammasome activation via P2X7³⁷. Lowering extracellular ATP levels in inflamed tissues can inhibit inflammation. Following AB4 treatment, GPI, TPI1, GAPDH, PGK1, PGAM1, ENO1, and PKM were downregulated, stimulating ATP breakdown to reduce inflammatory damage. LDHA and LDHB which were downregulated promote anaerobic metabolism of pyruvate with L-lactate and NADPH production³⁸. This results in less energy for the cow and is not conducive to fighting inflammation. LDHA and LDHB were upregulated with AB4 treatment, which enhanced the aerobic metabolism of pyruvate to provide energy for fighting against inflammation. In the meantime, GPI, G6PD, PGD, and TALDDO1 in the pentose phosphate pathway was upregulated in CM cows, leading to bulk NADPH production. Activated in nature by microbes and microbial-derived products, the phagocyte NADPH oxidase rapidly assembles and generates reactive oxidant intermediates (ROIs) in response to infectious threats³⁹. NADPH oxidase plays a key role in modulating inflammation and injury, distinct from its antimicrobial function³⁹. However, ROIs can directly injure cells by damaging DNA, proteins, and lipids⁴⁰. GPI, G6PD, PGD, and TALDDO1 downregulation with AB4 can prevent oxidative damage by excessive NADPH.

Leukocyte migration from the blood into the sites of infection is a vital immune surveillance strategy⁴¹. In cows with CM, Rac2 upregulation promotes leukocyte motility⁴²; RhoA and MLC upregulation indirectly promotes tail retraction⁴³; ERM, actin, and α-actin upregulation indirectly regulates the docking structure⁴⁴, and CLDN8 downregulation promotes leukocyte transendothelial migration. This is also responsible for the high SCC in mastitis milk. After AB4 treatment, Rac2, MLC, ERM, actin, and α-actin were downregulated, and leukocyte migration was inhibited. ITGB2 binds to JAM1, JAM3, and ICAM3, promoting transendothelial migration of neutrophils and T cells, and phagocytosis of apoptotic neutrophils by macrophages, respectively^45,46. ITGB2 was downregulated by AB4. These results indicate that AB4 has a great potential to reduce SCC.

Phagocytosis plays an essential role in host defense mechanisms through the uptake and destruction of infectious pathogens⁴⁷. After opsonization with antibodies (IgG), foreign extracellular materials are recognized by Fc gamma receptors. IgG is downregulated in mastitis cows, reducing the ability to recognize antigens, be caused by a vast array of immune evasion mechanisms of S. aureus. CFL1 plays a role in the regulation of cell morphology and cytoskeletal organization in epithelial cells⁴⁸. The Arp2/3 complex mediates the formation of branched actin networks in the cytoplasm, providing a force for cell motility⁴⁹. CFL1, ARPC4, and ARPC3 are upregulated during cow mastitis, promoting macrophage, neutrophil, and monocyte motility to destroy infectious pathogens^50,51. Following AB4 treatment, IgG was upregulated, whereas CFL1, ARPC4, and ARPC3 were downregulated, indicating that invasive pathogens were effectively controlled.

The most common pathway of mastitis infection is the complement and coagulation cascade pathway, also the main pathway for the host to deal with S. aureus³³. The complement system is a proteolytic cascade in blood plasma and a mediator of innate immunity, a nonspecific defense mechanism against pathogens. The main consequences of complement activation are the opsonization of pathogens, recruitment of inflammatory and immunocompetent cells, and direct killing of pathogens⁵². Pathogens have a greater effect on the blood-milk barrier, and consequently, a larger transfer of blood proteins to milk occurs in mastitis⁵³. Together with fibrinogen alpha (FGA) and fibrinogen beta (FGB), polymerize to form an insoluble fibrin matrix⁵⁴. FGA and FGB are upregulated with fibrin monomer production, further transfer to fibrin clots, causing deterioration in milk quality. Plasmin, which transfers fibrin clots to fibrin degradation products via the fibrinolytic system, reduced production due to the downregulation of PLG, HCII, F2, A1AT, and α2AP. C1q is the first subcomponent of the C1 complex in the classical pathway of complement activation⁵⁵. Functions in the lectin pathway of complement play a key role in innate immunity by recognizing pathogens through patterns of sugar moieties and neutralizing them. The lectin pathway is triggered upon the binding of mannan-binding lectin (MBL) and ficolin to sugar moieties, leading to activation of the associated proteases MASP1 and MASP2⁵⁶. Here, C1, MBL, MASP1, and MASP2 were downregulated in mastitis. The classical and lectin pathways of the complement cascade were inhibited, and AB4 treatment C1 and MBL were upregulated, activating the complement system. VTN and CLU protect cells against apoptosis and cytolysis by complement^57,58. Bacterial invasion into the mammary gland induced VTN and CLU downregulation, promoting cell apoptosis and cytolysis. Both VTN and CLU were upregulated after AB4 treatment. The membrane attack complex (MAC) forms trans–plasma membrane channels on the surface of pathogenic bacteria, causing cell lysis and death; only five of the complement system proteins eventually formed MAC subunits: one unit each of complement C5b, C6, C7, and C8 and several units of complement C9⁵⁹. C8A, C8B, C8G, and C9 were downregulated in mastitis, indicating that the host's ability to eliminate the bacteria is diminished. Following AB4 treatment, all proteins were restored to normal levels.

In summary, pathogenic bacterial infection upregulated PKM, LDHB, LDHA, ALDOA, PGD, GPI, and ALDOC, increased ATP production, which promotes leukocyte recruitment and NALP3-inflammasome activation, increases NADPH production, and promotes ROIs modulating inflammation and injury. These proteins were restored by AB4, which indicates that inflammation was inhibited. Invasion of pathogenic bacteria leads to transdermal migration of leukocytes into the mammary tissue, elevating somatic cells in the milk, increasing fibrinogen precursors, and decreasing plasmin production, resulting in fibrin clot deposition, eventually causing deterioration in milk quality. Bacterial infection of mammary tissue inhibits activation of the complement system, reduces MAC production, and protects pathogenic bacteria from killing through the complement immune system, thereby promoting apoptosis of mammary epithelial cells. Intramuscular infusion of AB4 can downregulate GPI, TPI1, GAPDH, PGK1, PGAM1, ENO1, PKM, GPI, G6PD, PGD, and TALDDO1 and restore LDHB and LDHA, reducing inflammatory damage caused by ATP and NADPH; downregulating Rac2, RhoA, MLC, ERM, actin, and α-actin ITGB2, inhibiting transendothelial migration of leukocytes, thereby reducing milk SCC; restoring PLG, FGA, FGB, reducing milk fibrin clots; upregulate C1, MBL, CLU, VTN, activating the complement system and reducing MAC, and directly inhibiting the invasion of pathogenic bacteria.

We investigated the differences in the milk proteomes of mastitis-affected cows following intramuscular AB4 injection. Our results suggested that AB4 may treat CM through multiple pathways by easing inflammation, reducing SCC, decreasing milk clots, and eradicating pathogenic bacteria. These results contribute to a better understanding of the AB4 mechanism in CM, providing theoretical support for its clinical application. Application of AB4 can reduce antibiotic usage, therefore, economic losses because of antibiotic residues in milk can be avoided, bacterial resistance will be controlled, and environmental pollution caused by antibiotic misuse will be reduced. Future research confirming the target proteins identified in this study of AB4 on dairy cows with CM is needed.

Funding

Our work has been possible thanks to the support of the National Key R&D Program of China (2018YFD0500900) and the Sichuan Department of Science and Technology Support Project (2019YJ0650). We would like to thank Editage (www.editage.cn) for English language editing.

Declaration of competing interest

All the authors declare that he/she has no conflict of interest.

Data Availability

Proteins’ GenInfo identifier (GI) accession numbers were converted into official gene symbol by uniport (https://www.uniprot.org/)-Bos-taurus-20191014.The full list of proteins identified in the milk whey samples are provided in the supplemental Tables S1. DEPs of T1/C1 and T1/T2 are provided in supplemental Tables S2. Tables S3 and Table S4 show enriched GO terms of DEPs. Table S5 lists significantly enriched KEGG pathways. The datasets generated during the current study are available in the Mendeley repository, Effect of anemoside B4 on milk whey in clinical mastitis-affected cows elucidated using tandem mass tag (TMT)-based quantitative proteomics - Mendeley Data.

Authors contributions

Shen Liu-hong: conceptualization and validation; Zhang Yue: writing-original draft and data curation; Shen Yu: formal analysis; Su Zhe-tong: supervision and software; Yu Shu-min: investigation; Cao Sui-zhong: funding acquisition; Zong Xiao-lan: project administration.

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Table 1. Eleven potential target molecular pathways of AB4.

Map ID	Map name	Test sequence number
Map ID	Map name	T1/C1	T2/C1
bta04610	Complement and coagulation cascades	18	8
bta00620	Pyruvate metabolism	4	3
bta04310	Wnt signaling pathway	4	3
bta00030	Pentose phosphate pathway	6	5
bta00010	Glycolysis / Gluconeogenesis	13	12
bta04066	HIF-1 signaling pathway	8	7
bta05230	Central carbon metabolism in cancer	5	4
bta04670	Leukocyte transendothelial migration	11	11
bta04810	Regulation of actin cytoskeleton	18	15
bta04666	Fc gamma R-mediated phagocytosis	7	5
bta05203	Viral carcinogenesis	10	8

No competing interests reported.

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Effect of anemoside B4 on milk whey in clinical mastitis-affected cows elucidated using tandem mass tag (TMT)-based quantitative proteomics

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