Rac1 signaling regulates LPS-induced mouse mastitis via inhibition of NLRP3, NF-κB and MAPK signaling pathways

Bovine mastitis characterized by mammary gland inammatory responses, is the most frequent and costly diseases in dairy cattle. Researchers have been seeking effective treatments for this disease, but still not very successful. Ras-related C3 botulinum toxin substrate 1 (Rac1) is implicated in various cellular functions, including apoptosis, ROS production and inammatory responses, and presents an attractive therapeutic target for many diseases. However, the effects of Rac1 signaling on mastitis remain unclear. The aim of this study is to investigate the effects of Rac1 signaling on mastitis via inhibition by Rac1 specic inhibitor NSC23766 based on a murine model of lipoplysaccharide (LPS)-induced mastitis. to


Results
The results revealed that NSC23766 signi cantly decreased the damage of mammary gland by LPS, reduced myeloperoxidase activities, and the productions of IL-1β, IL-6, TNF-α and MCP-1 gene expression in the mammary glands with LPS perfusion. Moreover, western blot analysis showed that NSC23766 inhibited the phosphoryation of p65, IκBα, ERK, and p38, and suppressed the expression of NLRP3.

Conclusion
These ndings suggested that administration of NSC23766 prevented the development of mastitis by inhibiting NLRP3, mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. Accordingly, this study may provide research basis for the development of new drugs against mastitis, and NSC23766 might be a potential therapeutic drugs for mastitis.

Background
Bovine mastitis, an in ammatory response in the mammary gland, is one of the most costly pathologies in the dairy industry [1]. Milk yield and milk quality are in uenced by this disease, and huge costs are paid on treatment and care, resulting more and more people paying attention on bovine mastitis. The main treatment for this disease is intramuscular or intravenous injection of antibiotics such as penicillin, streptomycin, and tetracycline. However, the excessive use of antibiotics contributes to the development of antibiotic-resistant pathogens. In large Chinese dairy herds, the prevalence of antimicrobial resistance in mastitis pathogens is comparatively high [2]. Thus, exploring alternatives to antibiotics in mastitis therapy is urgently needed.
Many pathogens are known to cause bovine mastitis, including bacteria, mycoplasma and yeasts [3]. The most common mastitis-causing bacteria includes Staphylococcus aureus and Escherichia coli [4], which invade the udder through the teat canal and cause a prompt in ammatory reaction [5].
Lipopolysaccharide (LPS) is a component of the outer membrane of gram-negative bacteria, and has been used to establish mouse model of mastitis [6]. LPS induces a potent in ammatory response in mammary tissue, which is similar to the natural mastitis. Pro-in ammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) are important in the development of LPS-induced mouse mastitis [7]. On the other hand, MAPK and NF-κB signaling pathways are known to regulate the expression of various pro-in ammatory genes, and play a crucial role in LPSinduced mouse mastitis [6]. Studies have revealed that mastitis are improved via inhibition of these two signaling pathways [8,9].
Ras-related C3 botulinum toxin substrate 1 (Rac1) is a member of Rac family of small guanosine triphosphatases (GTPase), and is known to regulate various cellular functions, such as cell proliferation and survival, migration and invasion, cellular adhesions, apoptosis, ROS and in ammatory responses [10][11][12][13]. Deregulation of Rac1 signaling is invovled in a number of human diseases, indicating Rac1 is an attractive therapeutic target. The inhibition of Rac activity shows anti-in ammatory effects in models of acute pancreatitis and sepsis [14,15]. Targeting Rac1 decreases paw swelling in early arthritis and to a lesser extent in chronic arthritis in collagen-induced murine model of rheumatoid arthritis [16]. NSC23766 is a Rac-speci c molecule inhibitor, and is often used to study the physiological functions of Rac1 [17].
Though previous study revealed that NSC23766 reduced the in ammatory cells in ltration and inhibited the expression of TNF-α and IL-1β [14], its detailed molecular mechanisms remain unknown. In this study, we aimed to investigate the effects and molecular mechanisms of NSC23766 in LPS-induced mastitis mice.

Materials And Methods
Animals and LPS-induced mouse mastitis model BALB/c mice, 6-8 weeks old (18 female and 9 male), were divided into three groups, and each group contained two female and one male raised in a cage. After suckling mice born on the 10th day, 18 female were randomly divided into three groups for next experiments. Group was used as control. Group was treated with LPS group. Group treated with LPS and NSC23766 group. The method of establishment of mastitis mouse model refers to our previous reports [5]. In brief, the teat duct of Group was infused with LPS (200 μg/mL). Group mice were intraperitoneally injected with NSC23766 (5 mg/kg, MedChemExpress) 1h before LPS (200 μg/mL) treatment. Group mice received an equal volume of PBS treatment. At 24 h after the LPS perfusion, mice were killed by CO 2 inhalation. The mammary glands were collected and stored at -80 °C for later analysis. All animal experiments were in line with Jilin University experimental animal management regulations, and the People's Republic of China Animal Protection Law.

Cell culture and treatments
The mouse mammary epithelial cell line (EpH4-Ev) were cultured in DMEM medium F12 (Hyclone) supplemented with 10 % fetal bovine serum (Clark) at 37°C with 5 % CO 2 . Cells plated into a 6-pore plate and pre-treated with NSC23766 (50, 100 and 200 μM) for 30 min. After that the cells were treated with LPS (5μg/mL) for 12 h, and collected for qRT-PCR and western blot analysis.

Histopathological Examination
The collected mammary tissues were xed in 10 % formalin for 48-72 h. Then xed mammary glands were dehydrated with graded alcohol, embedded in para n, and stained with hematoxylin and eosin (H&E). Finally, the histopathologic changes were observed by light microscopy.

Myeloperoxidase (MPO) Assay
In order to detect the activity of MPO in the mammary glands, the collected mammary glands were homogenized, and detected by MPO kit (Jiancheng Bioengineering Institute of Nanjing). The speci c experimental procedures were carried out according to the instruction.

qRT-PCR Analysis
Total RNA of mammary glands was extracted with Trizol (Invitrogen, USA), and complementary DNA (cDNA) was reversed transcription by the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scienti c, San Jose, CA). Primers were acquired from Sangon Biotech Co. Ltd (Shanghai, China, Table 1). The genes expressions of mammary glands were analyzed by using a 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The conditions were as follows: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.

Western blotting
Total protein in mammary tissue was extracted with the T-PER™ Tissue Protein Extraction Reagent (ThermoFisher, product number: 78510), which contains a propreitary detergent in 25 mM bicine and 150 mM sodium chloride. Protein concentration was determined by BCA protein assay kit. The samples were separated by 10 % SDS polyacrylamide gel, and then proteins were transferred to PVDF membranes. Membranes were blocked in 5 % bovine serum albumin for 2 h. Whereafter, the membranes were probed with primary antibody (1: 1000 dilution in TBST) overnight at 4 °C, washed thrice in TBST with gentle mixing, and then incubated with peroxidase-conjugated secondary antibody (1: 20000 dilution in TBST) for 3 h. After thrice washing in TBST, the samples were visualized with the ECLPlus Western Blotting Detection System (GE Healthcare, Chalfont St Giles, UK). Finally, the western blot bands were quanti ed by the software of ImageJ. The primary antibodies of ERK, P38, P65, Ikb, NLRP3 and GADPH were purchased from Boster bioengineering co. LTD (China).The primary antibodies of p-ERK (Thr202+Tyr204), p-p38 (Tyr182), p-p65 (ser536), p-iκb (S32/S36) were purchased from Bioworld Technology, Inc. (USA).

Statistical analysis
The data was analyzed by using GraphPad Prism 5.0 software. The western blot band intensities were quanti ed by the software of ImageJ. Difference among groups was analyzed by one-way analysis of variance (ANOVA) and the Tukey multiple comparison test. Values were expressed as the means the ± standard deviation (SD). The signi cance threshold was set at 0.05.

NSC23766 decreased LPS-induced pathologic changes in mastitis mice
As shown in Fig 1, LPS signi cantly induced clinical symptoms of mastitis, such as slight swelling, displayed redness ( Fig 1B) compared with control group (Fig 1A), but NSC23766 treatment (5 mg/kg) signi cantly decreased LPS-induced clinical symptoms of mastitis, which meant that NSC23766 possessed anti-in ammatory activities. To con rm the anti-in ammtory effects of NSC23766 on mastitis mice, histopathological examination was carried out. It was found that NSC 23766 also decreased LPSinduced pathologic changes including widened breast alveolar intervals, in ammatory cell in ltration and destruction of mammary acinar integrity (Fig 1D-1F), indicating its protective role in mastitis.

NSC23766 decreased LPS-induced MPO activities
Neutrophils are one of the rst cells recruited to sites of in ammation, and MPO is the most abundantly expressed enzyme in neutrophil. Next, we examined main type of in ammatory cell in ltration in mammary gland. It was showed that LPS signi cantly increased MPO activities compared to the control group (Fig 2), but pretreatment with NSC23766 (5 mg/kg) abolished this increase of MPO activity induced by LPS (Fig 2), indicating that neutrophils are the main cell type in ltrated in mammary gland of mastitis.

NSC23766 decreased LPS-induced cytokines and chemokines gene expression
Given that cytokines and chemokines play crucial role in initiating in ammatory response, here we examined the effect of NSC23766 on expression of IL-1β, IL-6, TNF-α and MCP-1 genes. As shown in Figure 3, IL-1β, IL-6, TNF-α and MCP-1 mRNA levels in the LPS group was markedly increased compared with control group. However, NSC23766 (5 mg/kg) pre-treatment also signi cantly decreased LPSinduced IL-1β, IL-6, TNF-α and MCP-1 mRNA levels in mammary glands of mastitis mice, implying that NSC23766 also plays a role in regulating these gene expression of IL-1β, IL-6, TNF-α and MCP-1 in mastitis.
NSC23766 inhibited LPS-induced the key proteins activities of NLRP3, MAPK, and NF-κB signaling pathways in mastitis To examine how NSC23766 regulated the gene expression of proin ammatory cytokines, we investigated several signaling pathways (NLRP3, MAPK, and NF-κB) involved in in ammation. Our results revealed that NLRP3 expression in the mammary tissues was dramatically increased by LPS perfusion compared to that of control group (Fig 4). Moreover, LPS resulted in signi cant phosphorylation of ERK and p38 in MAPK signaling pawthway (Fig 5), p65 and IκBα in NF-κB signaling pawthway (Fig 6). However, pretreatment with NSC23766 signi cantly inhibited the activities of NLRP3 and reduced phosphorylation of p65, IκBα, ERK, and p38 suggesting an important role of NSC23766 in regulating these signaling pathway to prevent LPS-induced in ammatory responses.
For further con rm the crucial role of NSC23766 in protecting LPS-induced in ammatory response in mastitis, mammary gland cells lines were used for next investigation. The results showed that LPS not only increased the expressions of IL-1α, IL-1β, IL-6, TNF-α and MCP-1 mRNA levels (Fig 7), but also induced the phosphorylation of p65 and IκBα in NF-κB signaling pathway (Fig 8). Surely, pretreatment with NSC23766 decreased LPS-induced these in ammatory response (Fig 7 and Fig 8). All above results suggest the key role of Rac1 signaling pathway in LPS-induced mastitis.

Discussion
Bovine mastitis, an in ammatory response of the mammary gland, is quite common in dairy cows [18]. Until now, mastitis still is a major challenge to the worldwide dairy industry. On the one hand, mastitis declines the production and quality of milk, which causes great economic losses [3]. On the other hand, mastitis can affect the fertility of cows [19]. For these reasons, it is crucial to nd an effective treatment program for mastitis.
Rac1 is one of Rho small GTPases, which is an important component in in ammatory reponse [20], and NSC23766 as a Rac-speci c inhibitor, exhibits signi cant anti-in ammatory effects in animal models of many diseases, such as acute pulmonary injury, colonic injury and septic lung injury [21][22][23]. In this study, we aimed to investigate the effects of NSC23766 on LPS-induced mastitis, and further explored its fundamental mechanisms.
Increasing publications suggest that Rac 1 plays a signi cant role in in ammatory reactions. Rac1 has special research value in the present study since data in the literature show that Rac1 regulates the progression of the in ammatory response, such as acute lung injury [24]. It was found that inhibition of Rac 1 activity with NSC23766 signi cantly weakened LPS-induced pathologic changes such as decreasing in ammatory cell in ltration, widened breast alveolar intervals and destruction of mammary acinar integrity. Moreover, we con rmed that inhibition of Rac 1 activity reduced MPO activity in mammary gland of mastitis, which is consistent with our previous research on the anti-in ammatory effects of baicalein [8]. MPO, a marker of neutrophil function and activation, is related to the neutrophil in ltration into the tissue and directly proportional to the number of neutrophils in the tissue [25].
Collectively, these results suggest that NSC23766 prevented LPS-induced in ammatory damage via suppressing neutrophil in ltration.
Cytokines are involved in the regulation of many local in ammations and systemic in ammation [26]. IL-1β, IL-6, TNF-α and MCP-1 are the most common cytokines and chemokines in several in ammatory reactions. To further investigate the protective role of NSC23766 in LPS-induced mastitis, the gene expression of IL-1β, IL-6, TNF-α and MCP-1 was measured. Compared with the LPS group, the levels of IL-1β, IL-6, TNF-α and MCP-1 in mammary gland were reduced by NSC23766 pretreatment. These results suggested that NSC23766 did attenuate the in ammatory reactions in LPS-induced mastitis, suggesting the important role of Rac1 signaling pawthway in mastitis.
Although we found that Rac1 is involved in LPS-induced mastitis, the signaling pathways in this process are still unclear. NLRP3, a component of the in ammasome, is composed of the NLRP3, ASC and caspase-1 [27,28]. Once NLRP3 in ammasome activated, it can convert the inactive pro-IL-1β to a biologically active form [29]. IL-1β is a pro-in ammatory cytokine which is the most important of all cytokines in the in ammatory process [30]. Moreover, the activation of NF-κB and MAPK signaling pathways are partly resulting in the activation of NLRP3 in ammasome [31]. In this study, it was found that NSC23766 signi cantly reduced LPS-induced the expression of NLRP3, indicating that NSC23766 inhibited pro-in ammatory cytokine IL-1β expression partly via in ammasome formation. Rac1 is a key mediator in a variety of cellular events, including ROS generation, chemotaxis, cytoskeleton remodeling and gene transcription [32]. It has been reported Rac 1 activates p38 MAPK signaling pathway via the p21 activated kinase 1 (PAK1) [33]. Moreover, MAPK signaling pathway activation increases pro-in ammatory cytokines secretions, which regulate the in ammatory response [34]. Our data indicated that NSC23766 pre-treatment signi cantly reduced ERK and p38 phosphorylation levels in mouse mammary gland, which is consistent with previous study. Since NSC23766 inhibits the MAPK pathway, we subsequently examined other more signaling pathways which are involved in the in ammatory response of mastitis. NF-κB, a main regulatory transcription factor, plays a crucial role in immune responses, such as the regulation of genes encoding pro-in ammatory cytokines [35]. Reports have shown that multiple drugs exhibit anti-in ammatory effects by inhibiting the NF-κB signaling pathway, such as cepharanthine, baicalein and morin [5,8,36]. Moreover, Rac 1 is closely related to NF-κB signaling pathway, which regulates the degradation of IκBα and nuclear translocation of STAT3-NF-κB complexes in starved cancer cells [37,38]. The results revealed that NSC23766 signi cantly inhibited the activation of p65 and degradation of IκBα in mouse mammary gland. These data indicated that NSC23766 exerted its antiin ammatory effects by inhibiting the activation of NLRP3, NF-κB and MAPK signaling pathways.

Conclusions
In summary, our work demonstrated the effects of NSC23766 as an inhibitor of Rac 1 in regulating LPSinduced mastitis in mice. Pretreatment with NSC23766 protected mammary gland from LPS-induced in ammatory responses via inhibiting the activation of NLRP3, NF-κB and MAPK signal pathways, implying an important role of Rac 1 signaling in regulating these processes. All data suggest that Rac1 is a key regulator in LPS-induced mastitis and provide a new understanding the role of Rac1 in the in ammatory responses process. Nevertheless, other more potential molecular mechanisms that may regulate LPS-induced mastitis in mice need to be further explored.

Availability of data and materials
Please contact corresponding author for data requests.
Authors' contributions CW wrote the paper and performed the experiments; AJ and WJ performed the experiments; XL and ZH analyzed the data; ZY and ZW contributed to design the experiments. All authors read and approved the nal manuscript.
Ethics approval and consent to participate All animal experiments were in line with Jilin University experimental animal management regulations, and the People's Republic of China Animal Protection Law.

Consent for publication
All authors have read the manuscript and approved the nal version.

Competing interests
The authors declare that they have no competing interests.         NSC23766 inhibited LPS-induced activities of NLRP3 signaling pathways in mastitis. Western blot analysis was performed for examining NLRP3 activities in mouse mammary gland. The results showed that NSC23766 signi cantly inhibited the activities of NLRP3 signaling pathways. The date is presented as mean ± SD (n = 5). P values of < 0.05 were considered signi cant. (***means P < 0.001).

Figure 5
NSC23766 inhibited LPS-induced the phosphorylation of ERK and p38 MAPK signaling pawthway in mastitis. Western blot analysis was performed for examining ERK and p38 activities in mouse mammary gland. The results showed that NSC23766 signi cantly inhibited the phosphorylation of ERK and p38 MAPK signaling pawthway. The date is presented as mean ± SD (n = 5). P values of < 0.05 were considered signi cant. (** means P < 0.01 and ***means P < 0.001).

Figure 5
NSC23766 inhibited LPS-induced the phosphorylation of ERK and p38 MAPK signaling pawthway in mastitis. Western blot analysis was performed for examining ERK and p38 activities in mouse mammary gland. The results showed that NSC23766 signi cantly inhibited the phosphorylation of ERK and p38 MAPK signaling pawthway. The date is presented as mean ± SD (n = 5). P values of < 0.05 were considered signi cant. (** means P < 0.01 and ***means P < 0.001).