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

Abstract 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.


| INTRODUC TI ON
Mastitis is a common infectious disease in humans and animals, causing significant economic losses in the dairy industry and affecting the quality and safety of dairy products. 1,2There are many causes of mastitis, and pathogenic microbial infection is one of the most common causes. 3Staphylococcus aureus (S. aureus) is one of the main pathogenic bacteria.Due to its characteristics of intracellular parasitize and immune escape, it is difficult to remove once invading the mammary gland. 4Therefore, S. aureus-induced mastitis is very difficult to cure.Currently, antibiotics are still the main treatment for mastitis caused by S. aureus in clinical practice, but the overuse of antibiotics will lead to drug residues and bacterial resistance.Besides, antibiotics had no significant effect on the bacteria clearance of mammary gland and the repair of the blood-milk barrier. 5Therefore, there is an urgent need to identify alternative therapeutic agents for the treatment of S. aureus-induced mastitis.
Saikosaponin A (SSA) is a triterpenoid saponin extracted from the medicinal plant Bupleurum falcatum. 6,7SSA has been reported to have various pharmacological activities, including ZHAO et al.
anti-inflammatory, 8 antioxidant 9 and anticancer effects. 10In primary mouse macrophages, SSA can inhibit LPS-induced TNFα and IL-1β production. 11SSA has also been demonstrated to alleviate CCL4induced acute hepatocellular injury by inhibiting oxidative stress and activation of inflammasome. 12In addition, SSA is considered to have a specific inhibitory effect on nuclear factor kappa B (NF-κB) activation. 9For example, SSA can significantly relieve neuropathic pain by inhibiting the NF-κB pathway and p38 MAPK activation in rats. 13It has also been shown that SSA alleviated hyperlipidemic pancreatitis by activating peroxisome proliferator-activated receptor γ (PPARγ) expression and inhibiting NF-κB inflammatory pathway. 14NF-κB signal pathway is also involved in the occurrence and development of mastitis.Ran et al. 15 found that dioscin improved LPS-induced mastitis by inhibiting NF-κB pathway and activating AMP-activated protein kinase (AMPK)/nuclear factor erythroid-2related factor 2 (Nrf2).Similarly, probiotic Enterococcus mundtii H81 inhibits the NF-κB pathway to mitigate mastitis caused by S. aureus in mice. 16However, whether SSA can ameliorate S. aureus-induced mastitis by inhibiting the NF-κB pathway remains to be further investigated.
Ferroptosis is a newly discovered form of cell death that is different from apoptosis, necrosis and autophagy. 17It is characterized by mitochondrial atrophy, lipid peroxidation, iron accumulation, heightened levels of prostaglandin endoperoxidase 2 (PTGS2) and reduced glutathione peroxidase 4 (GPX4). 17Ferroptosis has been implicated in various diseases, including cancer, 18 neurodegeneration 19 and inflammatory disease. 20Zhang et al. demonstrated that ferroptosis was involved in clinical mastitis in dairy cows and heme oxygenase 1 (HMOX1) promoted ferroptosis in mammary epithelial cells via FTH1. 21IL-6 promoted ferroptosis and inflammation through the Nrf2 signalling pathway in goat mammary epithelial cells. 22Besides, NF-κBp65 phosphorylation inhibited ferroptosis to alleviate ulcerative colitis. 23And ferrostatin-1 (Fer-1), a ferroptosis inhibitor, significantly reduced the level of toll-like receptor 4 (TLR4), phospho-nuclear factor kappa B (NF-κB) and phospho-inhibitor of kappa Bα (IκBα) in rats. 24Notably, the effect of SSA on ferroptosis and whether SSA can alleviate S. aureus-induced mastitis in mice by regulating ferroptosis remain unclear.
In this study, we found that SSA significantly ameliorated S. aureus-induced mastitis by inhibiting ferroptosis and inflammation.The results suggest that SSA may be a promising therapeutic agent for the prevention and treatment of S. aureus-induced mastitis, as well as other inflammatory diseases associated with ferroptosis.

| Animals
All BALB/c mice (21-25 g, 6-8 weeks old) were provided by Liaoning Changsheng.They were given plenty of water and food and placed in a specific pathogen-free facility with a 12-h light and dark cycle.
All mice experiments were conducted abide by and approved by the IACUC of Jilin University.To establish a S. aureus-induced mastitis model, 1 × 10 8 CFU/mL S. aureus 100 μL was injected into the udder canals of L4 (left) and R4 (right), and tissue samples were collected 24 h later.SSA (5, 10 and 20 mg/kg) was injected intraperitoneally 1 h before S. aureus treatment.The doses of SSA used in this study were based on previous study. 25

| Reagents
Saikosaponin A (purity > 98%) was obtained from Sigma.ELISA kits were purchased from BioLegend.The antibodies were obtained from Affinity Biosciences.GPX4 was gained from Bioss.EX-527 was purchased from Selleck Chemicals.

| Bacteria cultures
Staphylococcus aureus (ATCC35556) was obtained from ATCC. S. aureus was cultured in tryptic soy broth medium at 37°C 180 r/min for 8 h to reach the mid-log phase.

| Haematoxylin and eosin staining
The mammary samples were harvested and immobilized with 4% paraformaldehyde for more than 48 h.Then, these samples were embedded in paraffin to prepare 4 μm sections and stained with haematoxylin and eosin.Histopathological changes were observed by optical microscopy (Olympus), and the histological score was as previously described. 26

| ELISA
The content of proinflammatory cytokine in the mammary gland was detected by ELISA assay kit according to the manufacturer's instructions.The absorbance values were read at 450 nm and 570 nm by an automated enzyme standard instrument.

| Western blots analysis
Proteins were extracted using protein extract, and protein concentrations were measured by bicinchoninic acid method.The proteins (30 μg) were separated using 12% SDS-PAGE and then the proteins to a PVDF membrane following methanol treatment.The PVDF membranes were blocked in 5% skim milk and then incubated with specific primary and secondary antibodies.Finally, the proteins were visualized using an enhanced chemiluminescence solution and were detected with the ECL system.

| Measurement of myeloperoxidase, glutathione, malondialdehyde and iron
Total glutathione (GSH) in tissue lysates was measured with GSH detection kit according to the manufacturer's instruction.The content of myeloperoxidase (MPO) and malondialdehyde (MDA) in tissue was measured with detection kits according to the manufacturer's instruction.Total Fe and Fe 2+ release levels were determined using iron assay kit according to the manufacturer's instructions.

| Statistical analysis
All data are showed as the means ± SEM and analysed using one-way anova (Dunnett's t-test).The data are considered statistically significant at p < 0.05 or p < 0.01.

| Saikosaponin A alleviates Staphylococcus aureus-induced mammary histological injury
To investigate the therapeutic effects of SSA on S. aureus-induced mastitis in mice, we conducted a dose-dependent study using various doses of SSA (5, 10 and 20 mg/kg).The results showed that SSA treatment significantly alleviated S. aureus-induced mammary damage, which was mainly manifested as inflammatory cell infiltration and structure destruction (Figure 1).However, the protective effects of SAA on S. aureus-induced mammary histological injury were prevented by SIRT1 inhibitor EX-527 (Figure 1).

| Saikosaponin A alleviates Staphylococcus aureus-induced inflammatory response
MPO activity and inflammatory cytokine production were tested to assess mammary inflammatory level.As demonstrated in Figure 2, MPO activity, TNFα and IL-1β increased markedly in S. aureustreated mice.Likewise, SSA markedly reduced the elevated levels of inflammatory markers caused by S. aureus, including MPO, TNFα and IL-1β (Figure 2).However, the inhibitory effects of SAA on S. aureus-induced inflammation were prevented by SIRT1 inhibitor EX-527 (Figure 2).

| Saikosaponin A improves Staphylococcus aureus-induced blood-milk barrier injury in mice
[29] Therefore, we investigated whether SSA can repair blood-milk barrier damage induced by S. aureus.We found that the levels of TJ proteins including ZO-1, occludin and claudin-3 were reduced in S. aureus-treated group (Figure 3).However, SSA significantly reversed these changes (Figure 3).Taken together, these re- S. aureus-induced TJ injury were prevented by SIRT1 inhibitor EX-527 (Figure 3).

| Saikosaponin A improves Staphylococcus aureus-induced ferroptosis in mice
To confirm the effects of SSA on ferroptosis, we examined the protein expression levels of PTGS2 and GPX4.The results showed that S. aureus significantly increased the level of PTGS2 but decreased the level of GPX4 (Figure 4).Iron accumulation is one of the main characteristic of ferroptosis, so we examined the iron content in mammary gland.We observed that S. aureus treatment markedly increased the level of Fe 2+ compared with untreated group (Figure 5).Meanwhile, S. aureus-treated mice showed higher MDA expression and lower GSH expression (Figure 5) than untreated mice.These results suggested S. aureus could induce ferroptosis in mammary gland tissue.However, SAA markedly alleviated S. aureus-induced ferroptosis (Figures 4,5).Furthermore, the inhibitory effects of SAA on S. aureus-induced ferroptosis were prevented by SIRT1 inhibitor EX-527 (Figures 4,5).

| Saikosaponin A attenuates Staphylococcus aureus-induced NFκ B pathway in mice
We next investigated the role of NF-κB pathway in S. aureus-induced mastitis in mice.Western blotting was used to detect the activation of NF-κB pathway and found that S. aureus increased the protein levels of phosphorylated p65 and IκB (Figure 6).S. aureus activates the NF-κB pathway in mice.Treatment of SSA suppressed S. aureusinduced NF-κB activation markedly (Figure 6).However, the inhibitory effects of SAA on S. aureus-induced NF-κB activation were prevented by SIRT1 inhibitor EX-527 (Figure 6).

| Saikosaponin A inhibits Staphylococcus aureus-induced inflammation and ferroptosis by activating SIRT1/Nrf2 signal pathway
Nrf2 was involved in inflammation and ferroptosis.In this study, expression of SIRT1, Nrf2 and HO-1 was decreased by S. aureus.
Treatment of SSA increased the expression of SIRT1, Nrf2 and HO-1 in a dose-dependent manner (Figure 7).However, the upregulation of SAA on SIRT1, Nrf2 and HO-1 expression was prevented by SIRT1 inhibitor EX-527 (Figure 7).Meanwhile, we found the inhibition of SSA on inflammation and ferroptosis were reversed by SIRT1 inhibitor EX-527.These data suggested SAA inhibited S. aureus-induced mastitis by activating SIRT1/Nrf2 signal pathway.

| DISCUSS ION
Mastitis is a common infectious disease in dairy cows, causing significant economic losses in the dairy industry and affecting the quality and safety of dairy products. 30S. aureus is one of the major causative agents of mastitis in dairy cows. 31It has been shown that the NF-κB pathway is involved in the pathological process of S. aureusinduced mastitis. 32Moreover, the NF-κB pathway has been associated with ferroptosis. 33However, the role of SSA in ferroptosis and S. aureus-associated mastitis and the underlying mechanisms are still unknown.In this study, we found that SSA could alleviate S. aureusinduced mastitis in mice.SSA treatment improved blood-milk barrier integrity, reduced mammary gland damage and inhibited ferroptosis in mammary gland.Moreover, SSA treatment also inhibited the activation of the NF-κB pathway during S. aureus-induced mastitis.
These findings suggest that SSA could be a potential therapeutic agent for the treatment of S. aureus-induced mastitis.
Inflammation is the host's protective reaction to tissue dysfunction. 27Proinflammatory cytokines including TNFα and IL-1β are closely related to the development and progression of mastitis. 34,35-1β is produced in the early stages of infection and is thought to excessive accumulation of Fe 2+ will lead to Fenton reaction, which ultimately causes ferroptosis. 43In the present study, we found that SSA significantly upregulated the level of GPX4 and GSH and downregulated PTGS2 and MDA caused by S. aureus.Similarly, the content of total Fe 2+ decreased in the SSA + S. aureus group compared with control group.Hence, we speculated that SSA might inhibit ferroptosis, thus alleviating S. aureus-induced mastitis.
SIRT1 is a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase that regulates key metabolic processes including oxidative stress, ageing and apoptosis through the deacetylation of various substrates.Numerous studies have shown that nuclear factor erythroid 2-related factor 2 (Nrf2) is an important downstream target of SIRT1 signalling. 49It is a transcription factor responsible for regulating the redox balance and protective antioxidant activity in mammalian cells.Under pathological conditions, it can transfer to the nucleus, bind to antioxidant response elements (ARE) and drive the expression of its target genes, such as heme oxygenase 1 (HO-1).

F I G U R E 2
Effect of Saikosaponin A (SSA) on MPO activity and inflammatory cytokine production in mammary gland.The values presented are the mean ± SEM. # p < 0.01 is significantly different from control group; **p < 0.01 is significantly different from Staphylococcus aureus (S. aureus) group.
prevent excessive oxidation of lipids and proteins by scavenging hydroxyl radicals, singlet oxygen and superoxide anions, thereby playing an effective role in antioxidant and anti-apoptosis.Therefore, SIRT1/Nrf2/HO-1 is an important way to maintain redox balance in vivo.Recent studies demonstrated that SIRT1/Nrf2 signalling was involved in the regulation of ferroptosis.Therefore, the effects of SSA on Nrf2 signalling pathway were measured.We found SSA could activate SIRT1/Nrf2 signalling, and SIRT1 inhibitor could reverse the inhibition of SSA on inflammation and ferroptosis.In conclusion, our study demonstrates that SSA could alleviate S. aureus-induced mastitis in mice by inhibiting ferroptosis and inflammation via the SIRT1/Nrf2 pathway.These findings suggest that SSA may be a potential therapeutic agent for the treatment of S. aureus-induced mastitis.F I G U R E 5 Effect of Saikosaponin A (SSA) on glutathione (GSH), iron and MDA production in mammary gland.The values presented are the mean ± SEM. # p < 0.01 is significantly different from control group; **p < 0.01 is significantly different from Staphylococcus aureus (S. aureus) group.F I G U R E 6 Effect of Saikosaponin A (SSA) on NF-κB activation in mammary gland.The values presented are the mean ± SEM. # p < 0.01 is significantly different from control group; **p < 0.01 is significantly different from Staphylococcus aureus (S. aureus) group.

F I G U R E 7
Effect of Saikosaponin A (SSA) on SIRT1, Nrf2 and HO-1 expression in mammary gland.The values presented are the mean ± SEM. # p < 0.01 is significantly different from control group; **p < 0.01 is significantly different from Staphylococcus aureus (S. aureus) group.