INTRODUCTION

Mastitis is one of the most costly diseases of dairy cattle [1]. Inflammation of the mammary gland has negative physical, chemical, and biological consequences [2]. Many microorganisms, including the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli, can cause mastitis [3–6]. E. coli is a major cause of bovine mastitis, particularly around the time of parturition or during early lactation [7]. Lipopolysaccharide (LPS), a principal component of the outer membrane of Gram-negative bacteria, triggers mastitis in bovine and mouse models and induces the production of various pro-inflammatory cytokines. LPS is a key trigger of inflammation [8]. The innate immune system plays an important role in defense against invading pathogens. Mouse mammary epithelial cell (MMEC) cultures are widely used to study the capacity of cells to sense and respond to mastitis-causing bacteria [9]. Such MMECs are the first line of defense against mammary bacterial infection, recognizing molecules typical of microbes via Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) [10]. TLR4, a key PRR, binds and contacts downstream mitogen-activated protein kinases (MAPKs) and nuclear transcription factor-kappa B (NF-kB) via receptor dimerization [11]. Activated MAPKs and components of the NF-kB signaling pathway regulate inflammation by promoting the expression of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), and other pro-inflammatory cytokines [12, 13]. Because a serious inflammatory reaction can cause major tissue damage, medicines that inhibit the activation of the TLR4 signaling pathway are potential anti-inflammatory agents and may prevent tissue damage caused by mastitis [14].

Indirubin, a 3,20 bis-indole isomer of indigo, is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine derived from a mixture of plants including Indigofera tinctoria L., Isatis tinctoria L., Strobilanthes cusia Kuntze, and Cnidii fructus [15]. Indirubin exhibits anti-leukemic, anti-proliferative, and hepatoprotective properties and is also a strong anti-inflammatory agent [16–22]. In this study, we investigated the protective properties of indirubin against mastitis caused by a LPS-induced inflammatory response, and we elucidated the associated anti-inflammatory mechanisms.

MATERIALS AND METHODS

Chemical and Reagents

Indirubin (HPLC ≥ 98 %) was purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China) (Fig. 1). Fetal bovine serum (FBS) and 0.25 % trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). LPS (E. coli 055:B5), human epidermal growth factor (EGF), insulin, and transferrin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM/F12/1:1) was purchased from Thermo Fisher Biochemical Products Co., Ltd. (Beijing, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell counting kit, HiScript® II RT SuperMix for PCR (R212-01) and AceQ qPCR SYBR Green Master Mix (Q111-02) were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). Mouse IL-1β platinum ELISA kit with pre-coated plates was purchased from eBioscience (San Diego, CA, USA). Mouse IL-6 and TNF-α platinum ELISA kits with pre-coated plates come from BioLegend (San Diego, CA, USA). β-Actin (BA2305) was purchased from Wuhan Boster Biological Engineering Co., Ltd. (Wuhan, Hubei, China); PTGS2 (cyclooxygenase-2 (COX-2)) and anti-TLR4 antibodies were purchased from GeneTex, Inc. (San Antonio, TX, USA). NF-kB pathway sampler kit was purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). MAPK family antibody sampler kit and phospho-MAPK family antibody sampler kit were purchased from Arigo (Taiwan, China).

Fig. 1
figure 1

Chemical structure of indirubin.

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In Vivo Study

Experimental Design

All animal procedures were approved by the Animal Welfare and Research Ethics Committee of Huazhong Agricultural University. Mastitis was induced in the L4 and R4 abdominal mammary glands of the mice models through an inoculate of 50 μL of 0.2 mg/mL LPS, as described previously [23]. Briefly, mice were separated from their offspring approximately 2 h prior to anesthetization with pentobarbital sodium salt (0.25 g pentobarbital sodium salt dissolved in 50 mL phosphate-buffered saline (PBS), 10 mg per 20 g i.p.). Thirty-six Kunming pregnant mice were purchased from the Center for Animal Experiment and ABSL-3 Laboratory of WHU (Hubei, China) and randomly divided into six groups: control group, dimethyl sulfoxide (DMSO) group, LPS group, 25 μM indirubin and LPS group, 50 μM indirubin and LPS group, and 100 μM indirubin and LPS group. LPS groups were used as positive controls. Different concentrations of indirubin and 0.1 % DMSO were administered by intraperitoneal injection approximately 1 h before and 12 h after LPS instillation, respectively. The control and LPS groups were given equivalent volumes of PBS. Following the LPS challenge after 24 h, the mice were euthanized by CO2, and then mammary gland tissue was harvested and stored at −80 °C in a cell-frozen storage tube freezer.

Detection of Inflammatory Cytokine Levels

Each mammary gland tissue sample was weighed and homogenized with phosphate buffer (w/v, 1:9) on crushed ice and then ground using a tissue homogenizer. Tissue samples were centrifuged at 2000g for 40 min at 4 °C, the lipid was discarded, and the supernatant was collected and centrifuged again at 2000g for 20 min at 4 °C to remove any remaining lipid. The supernatant was used to detect IL-1β, IL-6, and TNF-α levels in the mammary gland tissue using ELISA following the kit manufacturer’s instructions.

Histopathologic Evaluation of Mammary Gland Tissue

Mammary gland tissues were harvested 24 h after the LPS challenge for histopathological examination. The tissue samples were fixed in 4 % paraformaldehyde for 48–72 h, dehydrated with graded alcohol, embedded in paraffin, and then stained with hematoxylin (H) and eosin (E).

In Vitro Study

Experimental Design

Indirubin was dissolved in DMSO at 5–15 mM, stored in aliquots at −20 °C, and further diluted (as appropriate) in a culture medium. Primary MMECs were isolated, cultured to 80–85 % confluence, and divided into the following groups:

  1. (1)

    Indirubin + LPS group (indirubin + LPS): MMECs were pretreated with indirubin at 25, 50, or 100 nM for 1 h and stimulated with LPS (1 μg/mL) for 24 h.

  2. (2)

    Indirubin group (indirubin): MMECs were cultured with 25, 50, or 100 nM indirubin for 24 h.

  3. (3)

    DMSO group (DMSO): MMECs were cultured with 0.1 % (v/v) DMSO for 24 h.

  4. (4)

    Control group (Con): MMECs were treated with a cell culture medium (vehicle control) of the same volume, for the same time, as the other groups.

  5. (5)

    LPS group (LPS): MMECs were stimulated with LPS (1 μg/mL) for 24 h as a positive group.

Primary MMEC Isolation and Culture

MMECs were isolated following a previously described method [24] with slight changes. In brief, the fourth and fifth mouse mammary tissues were aseptically harvested from the mice after 18 to 20 days of pregnancy. Tissue fragments were washed in PBS with 2 % (w/v) penicillin-streptomycin until the wash solutions were clear and then minced using surgical scissors, and the minced samples were digested for 3 h at 37 °C at 110 revolutions/min in an oscillating incubator. The digestion mix was 0.2 % (w/v) each of collagenases I and II in 45 mL DMEM/F12 and 5 mL FBS (sterilized by filtering through a 0.45-μm-pore-sized filter) and 0.25 % (w/v) trypsin-EDTA. Each digest was centrifuged at 250g for 5 min and filtered through a 70-μm-pore-sized nylon mesh (Falcon). Each filtrate was digested with 0.25 % (w/v) trypsin-EDTA and filtered through a 40-μm-pore-sized nylon Tamis cell strainer to remove epithelial organoids. Differential adhesion was used to remove fibroblasts, red blood cells, and endothelial cells (which attached to the tube walls at different times). Primary MMECs were cultured in the basic serum-free medium DMEM/F12 (1:1) containing 5 ng/mL EGF, 5 μg/mL insulin, 5 μg/mL transferrin, 10 % (v/v) fetal bovine serum, and 1 % (w/v) penicillin-streptomycin, at 37 °C under 5 % (v/v) CO2 in a humidified atmosphere.

MTT Assay of Cell Viability

The effect of indirubin on primary MMEC viability was measured using the standard MTT assay as previously described [14]. In brief, 1 × 104 MMECs were seeded into 96-well plates for 24 h, indirubin and LPS were added or were not added, and incubation was continued for an additional time of 24 h. Indirubin was dissolved in DMSO; however, the DMSO concentration in the assay was <0.1 %; 0.1 % (v/v) DMSO thus served as the control. MTT was added (to 5 % w/v) to each well, and the culture was continued for 4 h. The medium was removed, and the cells were washed three times with PBS. The formazan crystals were dissolved in 150 μL DMSO/well; the absorbance was acquired at 570 nm using a microplate reader.

Total RNA Extraction and qRT-PCR

Total RNA extraction and qRT-PCR followed as previously described [25] with slight changes. In short, MMECs (1 × 106) were seeded into six-well plates and grown until 80–85 % confluent. Indirubin and LPS were added or were not added, and incubation was continued for an additional time of 24 h. The cells were washed twice with ice-cold PBS; 1 mL of the TRIzol (Molecular Research Center, USA) reagent was added to each well and processed as directed by the manufacturer, and the cell lysates were collected. Genomic DNA was removed from all samples by treatment with 4× genomic DNA (gDNA) wiper mix, and RNA was reverse-transcribed into complementary DNA (cDNA) using the HiScript® II QRT SuperMix for qPCR (with the gDNA wiper). Relative mRNA concentrations were quantitated and subjected to qRT-PCR using the ViiA™ 7 system (Applied Biosystems), SYBR® Green Master Mix, and the platinum SYBR Green qPCR Super Mix with 6-carboxyl-X-rhodamine II (ROX II), following the manufacturer’s instructions (Vazyme Biotech Co., Ltd., Nanjing, China). The primers used are listed in Table 1 [26–29]. The PCR cycling conditions were 2 min at 50 °C followed by 2 min at 95 °C and then 40 cycles of 15 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C. Each reaction mixture contained 1 μL of cDNA, 5 μL of the SYBR Green Super Mix, and sense and anti-sense primers. Each sample was run three times in triplicate; the data were averaged. Melting curves were constructed to assess PCR accuracy. The 2−ΔΔCt method was used to measure the expression levels of calibrator genes. β-Actin served as an internal control. We calculated ΔCt values as follows: ΔCt = Ct (target gene) − Ct (housekeeping gene) and also the following: ΔΔCt = ΔCt (treatment) − ΔCt (control). Amplitude variation served as a surrogate measure of gene expression.

Table 1 Sequence of Primers Used in the Current Investigation in qRT-PCR
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Enzyme-Linked Immunosorbent Assay

MMECs (1 × 106) were seeded into six-well plates and grown until 80–85 % confluent; indirubin and 1 μg/mL LPS were added or were not added, and incubation was continued for an additional time of 24 h. Cell-free supernatants were subsequently used for the pro-inflammatory cytokine assays with mouse ELISA kits according to the manufacturer’s instructions.

Western Blotting

MMECs (1 × 106) were seeded into six-well plates and grown until 80–85 % confluent; indirubin and 1 μg/mL LPS were added or were not added, and incubation was continued for an additional time of 24 h. The cells were washed twice with ice-cold PBS. Total proteins were extracted via rapid lysis. Protein extraction reagent was added to lysates on ice, followed by centrifugation at 12,000g for 10 min to collect supernatants. The proteins were quantitated using the BCA protein assay. Equal amounts of protein (20–30 μg) were loaded onto a 10 % (w/v) SDS-polyacrylamide gel, electrophoresed, and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5 % (w/v) BSA (Bio-Sharp) with Tris-buffered saline (TBST) containing 0.05 % (v/v) Tween-20 at room temperature for 3 h and then washed three times with TBST for 10 min each time. Primary antibodies were diluted in TBST and incubated with the membrane overnight, shaking at 4 °C. Each membrane was then washed with TBST followed by incubation with an HRP-conjugated secondary antibody at room temperature for 1 h while shaking. Finally, each membrane was developed using an enhanced chemiluminescence (ECL) detection kit and visualized using a chemiluminescence detection system (ChemiDoc; Bio-Rad, USA). Band densities were calculated using ImageJ software (BioTechniques, NY, USA).

Statistical Analysis

All data are expressed as the means ± SEMs and were compared by one-way analysis of variance (ANOVA, Dunnett’s t test). A p value <0.05 was considered to reflect statistical significance.

RESULTS

Histological Analysis of Mammary Gland Tissue

Histological analysis is the most direct method for evaluating tissue injury and indirubin treatment effects. Pathological changes and inflammatory cells were negligible in the control (Fig. 2a) and DMSO (Fig. 2b) groups. However, in the LPS groups, the mammary alveolus was hyperemic and thicker than the alveolus space and neutrophil infiltration (Fig. 2c). The indirubin ameliorated LPS-induced pathological changes in a dose-dependent manner (Fig. 2d–f). Furthermore, we observed fewer neutrophils and macrophages in the alveolus space as well as reduced thickness of the mammary alveolus, especially in the 100-μM dose group.

Fig. 2
figure 2

Effect of indirubin on histological changes in LPS-induced mastitis mice. Different concentrations of indirubin (25, 50, and 100 μM) were given by i.p. injection 1 h before and 12 h after LPS administration. Mouse mammary glands (n = 3–5) from each experimental group were processed for histological evaluation at 24 h after LPS challenge. Representative histological changes of the mammary gland from different groups: a control group, b DMSO group, c LPS group, d indirubin (25 μM) and LPS group, e indirubin (50 μM) and LPS group, and f indirubin (100 μM) and LPS group, all intervals of 1 h (hematoxylin and eosin staining, magnification ×200).

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Detection of Inflammatory Cytokine Levels in the Mammary Gland Homogenates

The expression levels of inflammation cytokines (IL-1β, IL-6, and TNF-α) in mammary gland tissue homogenates were measured by an ELISA. LPS challenge caused a significant increase in all three pro-inflammatory mediators compared with the control group (all p values <0.001). The indirubin treatment inhibited the expression of IL-1β, IL-6, and TNF-α in the LPS-stimulated mastitis mouse models in a dose-dependent manner. Furthermore, all levels were significantly lower than those in the LPS groups (p values <0.001) (Fig. 3a–c).

Fig. 3
figure 3

Effect of indirubin on IL-1β (a), IL-6 (b), and TNF-α (c) in the mammary gland with LPS-stimulated mastitis. Mice were given intraperitoneal injection of indirubin (25, 50, and 100 μM) 1 h before and 12 h after LPS administration. Tissue homogenates were used to evaluate measures with ELISA. The values are presented as the means ± SEM of three independent experiments. ### p < 0.001, versus control group; *p < 0.05, **p < 0.01, and ***p < 0.001, versus LPS group.

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Effect of Indirubin on Cell Viability

The MTT assay was used to show that indirubin was not toxic to MMECs. Cell viability was not affected upon exposure to 0–100 nM indirubin, 0.1 % (v/v) DMSO (the maximal level present in the indirubin solution), or 1 μg/mL LPS, for 24 h (Fig. 4). Moreover, the order of addition of LPS and indirubin had no obvious effect on viability.

Fig. 4
figure 4

Effects of indirubin on the cell viability in MMECs. Primary MMECs were cultured with different concentrations of indirubin (25, 50, and 100 nM) in the absence or presence of 1 μg/mL LPS for 24 h. The cell viability was determined by MTT assay. The values are presented as the means ± SEM of three independent experiments.

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Indirubin Reduced the Levels of Pro-inflammatory Cytokines Induced by LPS

We used ELISA and qRT-PCR to measure the levels of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Compared with the control group, LPS significantly increased the levels of these cytokines (all p values <0.001). However, these increases were significantly abrogated by indirubin, in a dose-dependent manner (Figs. 5a–c and 6a–c).

Fig. 5
figure 5

Effects of indirubin on secretion of IL-1β (a), IL-6 (b), and TNF-α (c) by LPS-stimulated MMECs. Primary MMECs were pretreated with indirubin (25, 50, and 100 nM) for 1 h prior to LPS (1 μg/mL) addition; culture was continued for 24 h. The relative expression levels for IL-1β, IL-6, and TNF-α were measured by ELISA. Data are presented as the means ± SEM (n = 3). ### p < 0.001, versus control group; *p < 0.05, **p < 0.01, and ***p < 0.001, versus LPS group.

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Indirubin Inhibited LPS-Induced COX-2 Expression

The COX-2 levels in cell lysates were analyzed by qRT-PCR and western blotting. COX-2 expression was increased by LPS addition, compared with that of the control; indirubin reduced the LPS-induced COX-2 expression with the increased indirubin concentration (Fig. 7a–c).

Effect of Indirubin on TLR4 Expression

TLR4 is a key immune receptor that plays an important role in the regulation of LPS-induced inflammation. We used western blotting to assess the effect of indirubin on TLR4 expression. LPS significantly increased the TLR4 level compared with the control and indirubin-treated groups. TLR4 expression by LPS-exposed MMECs was inhibited by indirubin in a dose-dependent manner (Fig. 8a, b).

Indirubin Suppressed the LPS-Induced NF-kB Signaling Pathway

To explore whether the NF-kB pathway mediated indirubin-mediated inhibition of the inflammatory response, the NF-kB and IkBα protein levels were determined by western blotting. The extents of P65 and IkBα phosphorylation were significantly increased after LPS treatment but were reduced by indirubin pretreatment, in a dose-dependent manner (Fig. 9a–c).

Indirubin Suppressed LPS-Induced MAPK Pathways Activated by LPS

The MAPK pathways are controlled by the ERK, JNK, and p38 protein kinases. A western blot was used to determine how indirubin interfered with MAPK pathways; the effects of indirubin on phosphorylation of P38, ERK, and JNK were analyzed. Phosphorylation increased in LPS-stimulated MMECs. Indirubin pretreatment markedly inhibited the LPS-induced increases in P38, ERK, and JNK phosphorylation, in a dose-dependent manner (Fig. 10a–d).

DISCUSSION

Mastitis is characterized by mammary gland inflammation attributable to intramammary bacterial, mycoplasmal, algal, or fungal infection [30]. Antibiotics are frequently used to cure mastitis, but resistance to single or several antibiotics is now commonplace [31]. The abundant mammary epithelial cells are key components of the udder, producing many key milk components supporting the immune system and affording adequate nutrition [32]. LPS induces a large-scale production of pro-inflammatory cytokines, such as TNF-α, and may thus cause tissue damage [33]. In recent years, many traditional Chinese medicine (TCM) extracts have been shown to exert anti-inflammatory effects on mastitis models [34, 35]. Indirubin is one such material, suppressing TNF-induced NF-kB activation [36]. However, indirubin has not yet been tested in animals; this is the first report of the anti-inflammatory effects of indirubin in terms of LPS-induced inflammation [37]; we elucidated the anti-inflammatory mechanisms of indirubin.

Our animal experiment effectively reflects in vivo conditions and provides insights into the anti-inflammatory effects of indirubin on LPS-induced mastitis mouse models. Following the LPS challenge, we observed an increase in thickness of the mammary alveolus walls compared with the control group, and large numbers of inflammatory cells, such as neutrophils and macrophages, were present in the thickened mammary alveolus. When treated with indirubin, inflammatory symptoms were attenuated (Fig. 2d–f). Compared with the control group, LPS significantly increased the levels of the pro-inflammatory cytokines and was significantly reduced by indirubin in a dose-dependent manner (Fig. 3a–c). Therefore, indirubin has anti-inflammatory effects on LPS-induced mastitis mouse models.

Next, we confirmed that indirubin exerted anti-inflammatory effects on LPS-induced MMECs. Pro-inflammatory cytokines, such as IL-1β, IL-6, IL-10, and TNF-α, are key mediators of most acute and chronic responses to inflammatory diseases. TNF-α and IL-6, the principal proinflammatory cytokines, are involved in the pathophysiology of endotoxin-induced mastitis, increasing the expression levels of adhesion molecules and triggering the production of reactive oxygen species (ROS) [38–41]. Consequently, we first used ELISA and qRT-PCR to explore whether indirubin suppressed the production of IL-1β, IL-6, and TNF-α in LPS-stimulated MMECs. We next used the MTT assay to confirm that indirubin was not toxic to MMECs up to a concentration of 100 nM (Fig. 4). The DMSO level in this test was <0.1 % (v/v), and LPS at 1 μg/mL was also minimally cytotoxic to MMECs. The following experiments reflected these findings. Indirubin (25, 50, and 100 nM) dose-dependently inhibited IL-1β, IL-6, and TNF-α production without a difference with the sequence of indirubin and LPS in LPS-stimulated MMEC. Thus, the inhibitory effects of indirubin on IL-1β, IL-6, and TNF-α expression should reduce the inflammatory responses of MMECs (Figs. 5a–c and 6a–c).

Fig. 6
figure 6

Effects of indirubin on secretion of IL-1β (a), IL-6 (b), and TNF-α (c) by LPS-stimulated MMECs. Primary MMECs were pretreated with indirubin (25, 50, and 100 nM) for 1 h prior to LPS (1 μg/mL) addition; culture was continued for 24 h. The relative expression levels for IL-1β, IL-6, and TNF-α were measured by qRT-PCR. Data are presented as the means ± SEM (n = 3). ### p < 0.001, versus control group; *p < 0.05, **p < 0.01, and ***p < 0.001, versus LPS group.

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The COX-2 expression level and that of prostaglandin E2 (PGE2) serve as pro-inflammatory markers; these materials are interventional targets when treating mastitis [42]. One of the two well-studied isozymes of COX-2 synthesizes the pro-inflammatory PGE2 when exposed to LPS [43]. We thus assumed that COX-2 inhibition might explain the anti-inflammatory effect of indirubin. COX-2 expression increased significantly after LPS challenge; indirubin pretreatment inhibited this rise in a dose-dependent manner (Fig. 7a–c). Indirubin thus protected MMECs. Thus, indirubin significantly downregulated the synthesis of the pro-inflammatory mediators IL-1β, IL-6, TNF-α, and COX-2, attributable to inactivation of the NF-kB and MAPK signal transduction pathways triggering production of these materials [44–46]. We next use indirubin to pro-protect MMECs and examined the effects of indirubin on the TLR4, NF-kB, and MAPK signaling pathways.

Fig. 7
figure 7

Anti-inflammatory effects of indirubin on COX-2 expression in LPS-induced MMECs. a MMECs were cultured with different concentrations of indirubin (25, 50, and 100 nM) for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. COX-2 was analyzed by western blotting and qRT-PCR. b Densitometric analysis of the effects of indirubin on COX-2 expression. Proteins were analyzed by western blotting; β-actin served as an internal control. The values are presented as the means ± SEM (n = 3). c The relative expression levels for COX-2 were measured by qRT-PCR. Data are presented as the means ± SEM (n = 3). ### p < 0.001, versus control group; **p < 0.01 and ***p < 0.001, versus LPS group.

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Toll-like receptors play important roles in innate immunity; the receptors recognize many pathogens and their pathogenic components (PAMPs) and initiate the innate immune response [47, 48]. Activated TLRs are essential for effective defense against invading pathogens. LPS first activates TLR4 to trigger the downstream NF-kB signaling pathway that regulates cytokine production and the expression of many inflammatory genes [49]. We showed by western blotting that indirubin inhibited TLR4 expression in a dose-dependent manner (Fig. 8a, b). Thus, indirubin weakens inflammation-mediated signaling to a downstream signaling pathway.

Fig. 8
figure 8

Effects of indirubin on TLR4 expression in LPS-induced MMECs. a Cells were cultured with different concentrations of indirubin (25, 50, and 100 nM) for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. Proteins were analyzed by western blotting. b Densitometric analysis of the effects of indirubin on TLR4 expression. Proteins were analyzed by western blotting; β-actin served as an internal control. ### p < 0.001, versus control group; ***p < 0.001, versus LPS group.

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The major regulatory transcription factor NF-kB exists as homo- or hetero-dimers with P50 and P65 proteins bound to the inhibitor IkBα; dissociation of the complexes is stimulated by cytokines, free radicals, stress, ultraviolet light, oxidized low-density lipoprotein, and bacterial and viral antigens [50]. Once stimulated, IkBα kinase engages in phosphorylation of NF-kB P65 and ubiquitin-mediated degradation of this product via the proteasome pathway. Activated NF-kB enters the nucleus and induces the expression of numerous genes involved in innate and adaptive immune regulation, cell adhesion, inflammatory responses, and anti-apoptotic mechanisms [51, 52]. It is thus not surprising that NF-kB inhibition mitigates the inflammatory response. It is well known that various natural herbs affect anti-inflammatory responses by inhibiting NF-kB signaling [53, 54]. We thus explored whether indirubin inhibited NF-kB P65 phosphorylation and IkBα degradation. The LPS-induced P65 phosphorylation was inhibited by indirubin in a dose-dependent manner via inhibition of IkBα degradation (Fig. 9a–c). Thus, indirubin may inhibit IL-1β, IL-6, and TNF-α production by abrogating NF-kB action via blockade of IkBα degradation and P65 phosphorylation. Indirubin may thus be useful to inhibit inflammatory reactions.

Fig. 9
figure 9

Effects of indirubin on phosphorylation of IkBα and NF-kB p65 in LPS-induced MMECs. a MMECs were pretreated with different concentrations of indirubin (25, 50, and 100 nM) for 1 h and then stimulated with LPS (1 μg/mL) for 24 h; cell lysates were then harvested. Proteins were analyzed by western blotting. b Densitometric analysis of the effects of indirubin on phosphorylation of NF-kB p65. c Densitometric analysis of the effects of different concentrations of indirubin on phosphorylation of IkBα. The values are presented as the means ± SEM (n = 3). ### p < 0.001, versus control group; **p < 0.01 and ***p < 0.001, versus LPS group.

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The Myd88-dependent pathways are involved in LPS-induced inflammation. We examined the effects of indirubin on phosphorylation of P38, ERK, and JNK of the MAPK signaling pathway; this pathway controls cytokine synthesis and release during the inflammatory response. Upon activation of MAP kinases, transcription factors in the cytoplasm or nucleus are, in turn, activated, triggering the expression of target genes causing biological responses, including the expression of proinflammatory mediators [55]. We investigated the effects of indirubin on phosphorylation of the ERK, JNK, and P38 MAPKs in LPS-stimulated MMECs; LPS induced ERK, JNK, and p38 MAPK activation. Importantly, indirubin pretreatment significantly suppressed the phosphorylation of P38, ERK, and JNK and may thus inhibit the production of IL-1β, IL-6, and TNF-α, exerting an anti-inflammatory action (Fig. 10a–d).

Fig. 10
figure 10

Effects of indirubin on MAPK expression in LPS-induced MMECs. a Indirubin treatment group after pretreatment with different concentrations of indirubin (25, 50, and 100 nM) for 1 h, and the cells were stimulated with LPS (1 μg/mL) for 24 h and harvested lysates. Proteins were analyzed by western blotting. b Densitometric analysis results of the effect of indirubin on phosphorylation of JNK expression. c Densitometric analysis results of the effect of indirubin on phosphorylation of P38 expression. d Densitometric analysis results of the effect of indirubin on phosphorylation of ERK expression. The cellular proteins were analyzed by western blotting, and β-actin served as an internal control. The values are presented as the means ± SEM (n = 3). ### p < 0.001, versus control group; **p < 0.01 and ***p < 0.001, versus LPS group.

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CONCLUSION

In conclusion, we demonstrated that indirubin exerted anti-inflammatory effects on mice. Indirubin pretreatment attenuated mammary gland histopathological changes and reduced the expression levels of pro-inflammatory genes, including those encoding IL-1β, IL-6, TNF-α, and COX-2, by inhibiting TLR4 expression and suppressing phosphorylation of IkBα, NF-kB P65, JNK, ERK, and P38. Thus, indirubin may be useful to prevent LPS-induced mastitis.