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Baicalin inhibits Staphylococcus aureus-induced apoptosis by regulating TLR2 and TLR2-related apoptotic factors in the mouse mammary glands
European Journal of Pharmacology 723 (2014) 481–488
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Baicalin inhibits Staphylococcus aureus-induced apoptosis by regulating TLR2 and TLR2-related apoptotic factors in the mouse mammary glands Mengyao Guo, Yongguo Cao, Tiancheng Wang, Xiaojing Song, Zhicheng Liu, Ershun Zhou, Xuming Deng, Naisheng Zhang n, Zhengtao Yang n Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province 130062, People's Republic of China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 August 2013 Received in revised form 10 October 2013 Accepted 17 October 2013 Available online 7 November 2013
Baicalin, the major active constituent of the isolated root of Scutellaria baicalensis, is widely used in China and Southeast Asian countries. Evidence has indicated that baicalin has multiple biological activities, including anti-apoptotic properties. Mastitis is a severe problem in humans and other animals and is characterized by mammary gland cell apoptosis. Staphylococcus aureus (S. aureus) is the major pathogen that causes mastitis. This study was designed to evaluate the protective effects of baicalin on the mammary glands during S. aureus-induced mastitis. In the present study, a mouse model was infected with S. aureus to induce mammary gland injury. Baicalin treatment was administered between 6 and 24 h after infection. Toll-like receptor 2, p53, BAX, BCL-2 and caspase-3 expression were analyzed using qPCR and Western blotting. The results indicated that baicalin significantly attenuated pathological damage and cell death in the mammary glands. Further studies revealed that baicalin down-regulated the expression of Toll-like receptor 2 (TLR2) and the phosphorylation of p53 in the mammary glands after S. aureus-induced mastitis. Baicalin also promoted the expression of BCL-2 at the mRNA and protein levels but inhibited BAX and caspase-3 (CASP-3) cleavage. Baicalin inhibited apoptosis and had protective effects on mammary gland tissues during S. aureus-induced mastitis. These effects were displayed by reductions in TLR2 expression and p53 phosphorylation and the regulation of apoptosis-related factors (BCL-2, BAX and CASP-3) in mammary gland tissues. & 2013 Elsevier B.V. All rights reserved.
Keywords: Baicalin Staphylococcus aureus Mastitis Toll-like Receptor 2 Apoptosis
1. Introduction Mastitis, which is caused by infections of the mammary glands, poses a serious problem in humans and animals (Seegers et al., 2003). Mammary gland infections are caused by infectious bacterial pathogens (Carneiro et al., 2009). Gram-positive bacteria tend to cause mastitis, and present in the mammary glands for long periods of time, resulting in serious injury to mammary gland cells. The structure of breast tissue and lactation function are damaged, and the condition can sometimes even be fatal (Guo et al., 2013). Staphylococcus aureus is the major gram-positive pathogen, causing both community-acquired and nosocomial infections (Li et al., 2010a). To date, there is no efficacious treatment for S. aureus-induced mastitis. A mouse model of S. aureus-induced mastitis was first described by Chandler (1970). Anderson and Chandler further characterized this model in the mid-1980s (Anderson and Craven, 1984). Since 2000, S. aureus has n
Corresponding authors. Tel./fax: þ86 431 87835 140. E-mail addresses: [email protected] (N. Zhang), [email protected] (Z. Yang). 0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.10.032
been widely employed in studies using a mouse model of microbial mastitis. Thus, we chose to use this model in our study. Local innate immunity plays a key role in initiating and coordinating homeostatic and defense responses in the mammary glands. Once S. aureus infects the mammary glands, recognition of the infection through the activation of pattern recognition receptors (PRRs) is imperative for the initiation of the immune response in the mammary glands (Elazar et al., 2010). TLR2, a PRR receptor, is activated by peptidoglycan and lipoteichoic acid, which are major constituents of the cell wall pathogen-associated molecular pattern (PAMP) of gram-positive bacteria, including S. aureus (Whelehan et al., 2011). TLRs have been reported to increase in number and become activated in response to a bacterial challenge in the mammary glands (Petzl et al., 2008). Activation of TLR2 can trigger proapoptotic signals and cause cell death in various systems (Sabroe et al., 2005). Caspase activities were observed to increase significantly in TLR2 signaling activated cells (Li et al., 2009b). S. aureus has been reported to induce apoptosis in epithelial cells, lymphocytes, and macrophages (Kahl et al., 2000; Tucker et al., 2000), and TLR2 expression was observed to be upregulated after S. aureus infection (Zhao et al., 2008).
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2.2. Histological analyses and apoptosis detection
Fig. 1. Chemical structure of baicalin.
Baicalin (Fig. 1), the major active constituent of the isolated root of Scutellaria baicalensis, is widely used in China and Southeast Asian countries (Ma et al., 2009). Evidence has indicated that baicalin has multiple biological properties, including antiapoptotic (Cheng et al., 2012), anti-oxidant (Cao et al., 2011), anti-tumor, anti-ischemic, anti-inflammatory (Guo et al., 2013) and immune system modulatory activities (Li et al., 2010b). Baicalin has been reported to play a beneficial role in various experimental models of invasive toxicants by alleviating inflammatory injury (Kim and Lee, 2012) via the involvement of TLR2- or TLR4-mediated innate immune reactions (Hou et al., 2012; Li et al., 2012). In our previous study, baicalin was demonstrated to play an anti-inflammatory role in S. aureus-induced mastitis (Guo et al., 2013). The present study was conducted to determine the antiapoptotic effects of baicalin on the mammary glands in a mouse model of S. aureus-induced mastitis and to examine the mechanisms of action involved.
2. Materials and methods 2.1. Animal experimental groups and drug administration In total, 60 adult female BALB/c mice (6–8 weeks old, weighing 40–45 g) were used in the present study. The mice were provided by the Experimental Animal Center of Norman Bethune Medical College, Jilin University, China. All procedures were conducted according to the US NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Jilin University. Fifty milliliters of culture aliquots was centrifuged and washed with phosphate-buffered saline (PBS) prior to resuspension. S. aureus were resuspended in 1000 μl PBS (2 108 CFU per 30 μl). A 100 μl suspension was used per breast. The S. aureus suspension was injected via teat canal for inducing infection in the mammary gland. Baicalin was dissolved in physiological saline. Within the first 24 h after infection was induced in the mammary glands, baicalin was intraperitoneally injected four times (at 6, 12, 18 and 24 h). The mice were divided into three groups as follows (1) Microbionation group (MG): The mouse model of S. aureusinduced mastitis was established and the mice were treated with the vehicle control (saline). (2) Baicalin administration groups (BAGs): The mouse model of S. aureus-induced mastitis was established, and the mice were intraperitoneally administered baicalin at 25, 50, or 100 mg/kg (Guo et al., 2013). The dosage of baicalin was determined based on the results of a previous study. (3) Control groups (CGs): The mice were treated with normal saline (as a vehicle control) at the same volume and time points as baicalin treatment.
Mammary gland tissues were fixed in 10% formalin for two weeks and obtained from embedded paraffin samples. The tissues were deparaffinized with xylene and rehydrated using graded alcohol for the staining analyses. The sections were stained with hematoxylin and eosin (H&E) and then visualized using a microscope (Olympus, Japan). Apoptosis was identified in the mammary gland tissue sections using the in situ terminal deoxynucleotidyl transferase (TdT)mediated deoxyuridine triphosphate (dUTP) biotin nick-end labeling (TUNEL) technique with the In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany). Paraffin wax-embedded tissue sections were treated with proteinase K without DNase and incubated at 37 1C with the terminal TdT/ nucleotide mixture for 1 h. The reactions were then stopped, and the samples were rinsed with phosphate-buffered saline (PBS). Nuclear labeling was developed using horseradish peroxidase and diaminobenzidine. Hematoxylin was used for counterstaining. The evaluation of the apoptotic indices was performed by detecting positively stained nuclei at 400 magnification. 2.3. Quantitative real-time polymerase chain reaction Total RNA was isolated from the tissue samples (50 mg tissue; n¼ 3/ treatment group) using the TRIzol reagent according to the manufacturer's instructions (Invitrogen, China). The concentration and purity of the total RNA were determined spectrophotometrically at 260/280 nm. First-strand cDNA was synthesized from 5 μg of total RNA using oligo(dT) primers and Superscript II reverse transcriptase, according to the manufacturer's instructions (Invitrogen, USA). Synthesized cDNA was diluted five times with sterile water and stored at 80 1C. The Primer Premier software (PREMIER Biosoft International, USA) was used to design specific primers for TLR2, p53, BCL-2, BAX, CASP-3 and β-actin based on known sequences (Table 1). Quantitative real-time PCR was performed on an ABI PRISM 7500 Detection System (Applied Biosystems, USA). Reactions were performed in a 25-μl reaction mixture containing 12 μl of 2 SYBR Green I PCR Master Mix (TaKaRa, China), 2 μl of diluted cDNA, 0.5 μl of each primer (10 μM), 0.8 μl of 50 ROX reference Dye II, and 9.2 μl of PCR-grade water. The PCR procedure for TLR2, p53, BCL-2, BAX, Caspase-3 and β-actin consisted of 95 1C for 30 s, followed by 35 cycles of 95 1C for 15 s, 63 1C for 30 s and 60 1C for 30 s. A melting curve analysis showed only one peak for each PCR product. A dissociation curve was run for each plate to confirm the production of a single product. The amplification efficiency for each gene was determined using the DART-PCR program (Peirson et al., 2003). The mRNA relative abundance was calculated according to the method of Pfaffl (2001), accounting for genespecific efficiencies and was normalized to the mean expression of β-actin. Results (fold changes) were expressed as 2-ΔΔCt in which ΔΔCt ¼ (CtTLR2/p53/BCL-2/BAX//CASP-3-Ctβ-actin)t (Ct TLR2/p53/ BCL-2/BAX//CASP-3 Ct β-actin) c, where Ct TLR2/p53/BCL-2/BAX/CASP-3 and Ctβ-actin are the cycle thresholds for TLR2/p53/BCL-2/BAX/CASP-3 and β-actin genes in the differently treated groups, respectively, t is the treatment group, and c is the control group. 2.4. Western blot analyses Mammary gland tissues were weighed and homogenized with phosphate-buffered saline (w/v: 1/9) on ice, and the total protein was then extracted according to the manufacturer's recommended protocol (Invitrogen, Beijing, China). The protein concentrations were determined using the BCA Protein Assay Kit. Samples with equal amounts of protein (50 μg) were fractionated on 10% SDS
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Table 1 Oligonucleotide primers used for qPCR. Name
TLR2 p53 BAX BCL-2 CASP-3 β-Actin
Primer sequence
Product size (bp)
Sense: 5'- TGTGCCACCATTTCCACG-3' Anti-sense: 5'-AAAGGGCGGGTCAGAGTT-3' Sense: 5′-GGGAATAGGTTGATAGTTGTC-3' Anti-sense: 5′-CAGGCTTTGCAGAATGGA-3' Sense: 5′-AGTCCTGCGGGGCGGAGGCCATGTT-3' Anti-sense: 5′-AACATGGCCTCCGCCCCGCAGGACT-3' Sense: 5′-CTACCGTCGTGACTTCGC-3' Anti-sense: 5′-GGGTGACATCTCCCTGTT-3' Sense: 5′-AATCTGACGGTCCTCCTG-3' Anti-sense: 5′-TCGCCAAATCTTGCTAAT-3' Sense: 5′-TAAAACGCAGCTCAGTAACAGTCGG-3' Anti-sense: 5′–TGCAATCCTGTGGCATCCATGAAAC-3'
188 181 194 184 198 182
Fig. 2. Histopathology of mammary tissue after infecting with S. aureus ( H&E staining 100 ). (A) Mammary tissues of CG. (B) Microbionation group (MG). ((C)–(E)) Baicalin groups, administered at 25, 50, or 100 mg/kg.
polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and blocked in 5% skim milk in PBST for 1.5 h at 25 71 1C. The membranes were then incubated at 4 1C overnight with 1:1000 dilutions (v/v) of the primary antibodies. The primary antibodies were including TLR2 (cat number GTX13855, GeneTex, Inc. USA, clone 24088), p53 (cat number 2524s Cell Signaling Technology, Inc. USA, clone 1C12 ), p-p53(cat number 9284s Cell Signaling Technology, Inc. USA, clone Ser15 ), BCL-2 (cat number 611902, Biolegend Inc. San Diego, USA, clone Poly6119), BAX(cat number 625102, Biolegend Inc. USA, clone Poly6251), CASP-3 (cat number sc-136219, Santa Cruz Biotechnology Inc. USA, clone 8B1.1), cleaved CASP-3 (cat number GTX86951, GeneTex, Inc., USA, clone Asp175) and β-actin (cat number KM9001, Sungene Biotech Co., Ltd, China, clone 6G3). After washing the membranes with PBST, incubations with 1:5000 dilutions (v/v) of the secondary antibodies were conducted for 1.5 h at 25 71 1C. The secondary antibodies were HRP-conjugated goat–mouse antibodies (cat number LK2003L, Sungene Biotech Co., Ltd, China). (name, supplier, cat number, clone, etc.) Protein expression was detected using an Enhanced Chemiluminescence Detection System. β-Actin was used as a loading control.
2.5. Data analyses Statistical analyses were performed using the SPSS software package (ver. 13 for Windows; SPSS Inc., Chicago, IL, USA). Significance was determined using a one-way ANOVA with a significance level of P o0.05. The data were assessed using the Tukey–Kramer method for multiple comparisons. All values are expressed as the means 7 S.D.
3. Results 3.1. Histopathological changes Mammary gland tissues were harvested 24 h after inducing infection and baicalin treatment. Pathological changes were not observed in the CG (Fig. 2A). In the MG (Fig. 2B), the lobules of the mammary glands were incomplete (mammary gland alveolar congestion and marked thickening of the alveolar walls were observed, the acini of the mammary glands were damaged, and the epithelial cells were destroyed). Many inflammatory cells,
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including neutrophils and macrophages, were observed in the mammary gland tissues. However, these histopathological changes were ameliorated in the BAG following treatment with baicalin doses of 25, 50 and 100 mg/kg (Fig. 2C–E). Although some inflammatory cells were observed among the mammary gland tissues, the lobules were complete and the acini were not destroyed.
3.2. Apoptosis detection Apoptotic mammary gland cells were detected using the TUNEL assay. Apoptosis was not observed in the CG (Fig. 3A). Apoptosis was significantly increased in the MG (Fig. 3B) compared to the CG and BAGs. The majority of the apoptotic mammary gland cells were found in the breast tissues that exhibited structural failures. Baicalin significantly reduced apoptosis in the BAGs compared to the MG, and apoptosis was ameliorated in the BAG following treatment with doses of 25, 50 and 100 mg/kg (Fig. 3C–E).
3.4. Baicalin suppressed p53 expression in the mammary glands during S. aureus-induced mastitis p53 is a key signaling molecule during the development of apoptosis. Ordinarily, p53 is phosphorylated to promote apoptosis. The mRNA expression levels of p53 were measured using quantitative RT-PCR, and the protein expression levels of p53 and p53 phosphorylation were measured using Western blot analyses (Fig. 5). p53 mRNA levels were significantly increased (P o0.05) in the MG and baicalin-administered groups compared to the CG. However, the p53 mRNA levels of the MG group did not differ from those in the baicalin-administered groups (at doses of 25, 50, or 100 mg/kg) in a statistically significantly manner (P 40.1). The protein levels of phosphorylated p53 were significantly increased in the MG and were suppressed by baicalin treatment (Fig. 5A). Phosphorylated p53 protein levels were decreased in the baicalinadministered groups compared to the MG. The protein expression levels of unphosphorylated p53 protein did not differ between the CG, MG and baicalin-administered groups. 3.5. Effects of baicalin on BAX, BCL-2 and CASP-3 expression in the mammary glands during S. aureus-induced mastitis
3.3. Effects of baicalin on TLR2 expression in the mammary glands To determine the effects of baicalin on TLR2 expression in S. aureus-induced mastitis, the mRNA and protein expression levels of TLR2 were measured using quantitative RT-PCR and Western blot analyses. The results are shown in Fig. 4. Stimulation with S. aureus led to robust increases in TLR2 mRNA and protein expression levels. These effects were significantly inhibited by baicalin treatment. The mRNA and protein expression levels were significantly increased (P o0.05) in the MG and were slightly elevated in the baicalin-administered groups compared to the levels in the CG. The levels were significantly reduced in the baicalin-administered groups compared to the levels in the MG. As the dose of baicalin increased, the effects tended to become more obvious. The results are shown in Fig. 4.
CASP-3 activation is well-established to play a crucial role in the apoptotic response. BAX and BCL-2 play common roles in regulating CASP-3 activation or suppression. We investigated whether BAX, BCL-2 and CASP-3 were affected by baicalin treatment in S. aureus-induced mastitis. The results are shown in Fig. 5. BAX expression was significantly increased at both the mRNA and protein levels in the MG (Fig. 6). BAX mRNA levels were reduced in the baicalin-administered groups at doses of 25, 50 and 100 mg/kg, and BAX protein levels were also significantly reduced in the baicalin-administered groups (Fig. 6A). BCL-2 protein and mRNA expression were significantly reduced in the MG compared to the CG, but BCL-2 expression was significantly increased in the baicalinadministered groups (Fig. 6). CASP-3 mRNA levels were significantly increased in the MG and BAGs (Fig. 6). The mRNA levels of CASP-3 did not differ from those in the MG in the baicalin-administered
Fig. 3. Apoptosis detection of mammary tissue after S. aureus infection (TUNEL staining 100 ). (A) Mammary gland tissues in the CG. (B) Mammary gland tissues in the microbionation group (MG). ((C)–(E)) Mammary gland tissues in the baicalin-treated groups (25, 50, or 100 mg/kg). The nucleus of apoptotic cells were indicated brown staining, showed by arrows.
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Fig. 4. Effects of baicalin on TLR2 in the mammary glands. (A) TLR2 protein levels in mammary tissue. (B) TLR2 mRNA levels in mammary tissue. Western blotting was performed to detect TLR2 protein levels in the control group (CG), the microbionation group (MG), and the baicalin administration groups (25, 50, or 100 mg/kg). qPCR was performed to detect the mRNA levels of TLR2. β-Actin was used as a control. The values are presented as the means 7 S.E.M. (n¼ 10). nPo 0.01 indicates a significant difference from the CG. # Po 0.01 indicates a significant difference from the MG.
groups at doses of 25, 50 and 100 mg/kg. CASP-3 protein expression was also significantly reduced, but cleaved CASP-3 protein levels were significantly increased in the MG. CASP-3 protein levels were not reduced in the baicalin-administered groups at doses of 25, 50 and 100 mg/kg, but cleaved CASP-3 protein levels were significantly reduced (Fig. 6A). As the dose of baicalin increased, the effects on BAX, BCL-2 and CASP-3 expression tended to become more obvious.
4. Discussion Mastitis, which is mainly caused by microbial infections, is a severe problem in humans and other animals (Seegers et al., 2003). There are two types of mastitis: clinical and subclinical (Kim et al., 2011). The mouse model of S. aureus-induced mastitis is the current standard (Notebaert and Meyer, 2006). In our previous study, we were able to successfully establish this model (Guo et al., 2013), and this model was also used in the present study. Histopathological changes were observed in the present study. In the MG, the lobules of the mammary glands were damaged and incomplete (the alveolar glands were congested, marked thickening of the alveolar walls was observed, and the epithelial cells were destroyed) (Fig. 2B). These observations were likely due to
S. aureus affecting the mammary tissue epithelial and endothelial cells, which is consistent with the results of previous research (Anaya-Lopez et al., 2006). Some inflammatory cells, including neutrophils and macrophages, were observed in the mammary gland tissues in the MG. These results were similar to those of our previous study (Guo et al., 2013). Apoptosis was significant in the MG (Fig. 3B). Most of the apoptotic mammary gland cells were found in the breast tissues that exhibited structural failures. Baicalin protected mammary tissue epithelial and endothelial cells and reduced mammary damage (Fig. 2C–E). Mammary gland injury was ameliorated in the baicalin-administered groups. Apoptosis was significantly ameliorated in the BAGs following the administration of doses of 25, 50 and 100 mg/kg (Fig. 3C–E). These results indicated that mammary gland tissues that had been infected by S. aureus were protected by baicalin treatment. In previous studies, TLR2, a key immune receptor that is a member of the TLR family, was observed to be activated by S. aureus infection in the mammary glands, and TLR2 expression was increased following infection (Yang et al., 2008). In the present study, TLR2 mRNA and protein levels were assessed and were found to be significantly increased in the MG compared to the baicalin-administered groups. The results of the present study were similar to those of the previous study. The S. aureus cell wall components peptidoglycan and lipoteichoic acid have been found
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Fig. 5. Effects of baicalin on p53 expression. (A) p53 protein levels in mammary tissue. (B) p53 mRNA levels in mammary tissue. Western blotting was performed to detect p53 protein levels and p53 phosphorylation in the control group (CG), the microbionation group (MG), and the baicalin administration groups (25, 50, or 100 mg/kg). qPCR was performed to detect the mRNA levels of p53. β-Actin was used as a control. The values are presented as the means 7S.E.M. (n¼ 10). nPo 0.01 indicates a significant difference from the CG. # Po 0.01 indicates a significant difference from the MG.
to activate TLR2 (Li et al., 2009a). Activation of TLR2 signaling mediates immune cell activation and cell death in various systems (Sabroe et al., 2005). In the present study, TLR2 mRNA and protein levels were reduced in the baicalin-administered groups. TLR2 overexpression has been reported to result in cellular susceptibility to apoptosis (Fan et al., 2005). Thus, our findings indicated that baicalin treatment had an anti-apoptotic effect, reducing the expression of TLR2 in the mammary glands following S. aureus infection. p53 is defined as a tumor suppressor and nuclear transcription factor and can regulate several major cellular functions, including the cell cycle, senescence, and cell death (Culmsee and Mattson, 2005). Previous studies have reported that p53 may mediate apoptosis that is induced by a range of insults, including DNA damage, hypoxia, withdrawal of trophic support, hypoglycemia, oxidative stress, and viral infections (Morrison et al., 2003). In the present study, p53 mRNA levels were significantly increased (Po0.05) in the MG and baicalin-administered groups. These results indicated that p53 could be activated by S. aureus infections in the mammary glands. p53 mRNA levels in the baicalin-administered groups did not significantly differ (P40.1) from those in the MG. However, the protein levels of phosphorylated p53 were suppressed by baicalin treatment (Fig. 4A). Phosphorylated p53 protein levels were decreased in the baicalinadministered groups compared to the MG. This result indicated that
baicalin had anti-apoptotic effects in mammary gland cells by suppressing p53 phosphorylation. Activation of TLR2 also triggers the activation of proapoptotic signals, including caspase activity (Yin et al., 1997). The results of the previous study indicated that CASP-3 activity increased significantly in TLR2 signaling activated cells (Fan et al., 2005). p53 plays roles in both the intrinsic and extrinsic apoptotic pathways. Activation of p53 results in the expression of proapoptotic members of the Bcl-2 family (Miyashita and Reed, 1995; Oda et al., 2000) and death receptors (Muller et al., 1998). The results of the previous study revealed that the proapoptotic protein Bax was activated by p53 phosphorylation and binding to the antiapoptotic protein Bcl-2, which resulted in the induction of apoptosis (Chipuk et al., 2004). To further characterize the nature of the protective effects of baicalin against apoptosis, we examined the effects of baicalin on BAX, BCL-2 and CASP-3 expression. Ordinarily, BCL-2 mediated the activation and translocation of BAX to the mitochondrial outer membrane (Chipuk and Green, 2006). BAX and BCL-2, which act as death agonists and antagonists, respectively, are two opposing proteins (Kroemer, 1997). Apoptosis occurs as a result of competing dimerization between the two proteins, the relative proportion of which ultimately controls the sensitivity or resistance of cells to apoptotic stimuli (Reed, 1994). Our results indicated that BAX mRNA and protein
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Fig. 6. Effects of baicalin on the activation of apoptosis-related factors. (A) BCL-2, BAX, and CASP-3 protein levels in mammary tissue. (B) BCL-2, BAX, and CASP-3 mRNA levels in mammary tissue. Western blotting or qPCR was performed to detect BCL-2, BAX, and CASP-3 protein or mRNA levels in the control group (CG), the microbionation group (MG), and the baicalin administration groups (25, 50, or 100 mg/kg). β-Actin was used as a control. The values are presented as the means 7S.E.M. (n ¼10). nPo 0.01 indicates a significant difference from the CG. # P o0.01 indicates a significant difference from the MG.
expression were increased in the MG and were reduced in the baicalin-administered groups. However, BCL-2 mRNA and protein levels were significantly increased (P o0.05) in the baicalinadministered groups compared to those in the MG. The relative proportion (BAX/BCL-2) was also higher in the MG than in the baicalin-administered groups. This result indicated that S. aureus induced apoptosis in the mammary glands, which was suppressed by baicalin treatment, and was consistent with the histopathological changes that were observed. CASP-3 is a major proapoptotic element. TLR2 can trigger CASP-3 activation and cause cell death and apoptosis (Harburg et al., 2007). BAX and BCL-2 also play common roles in regulating CASP-3 activation or suppression (Shi et al., 2010). In the present study, we observed striking effects on CASP-3 mRNA or protein expression levels following baicalin treatment. CASP-3 mRNA levels and cleaved CASP-3 bands (19 Kd and 17 Kd bands) were significantly increased in the MG (Fig. 5). The mRNA levels of CASP-3 were not influenced by baicalin treatment. The cleaved CASP-3 proteins were significantly reduced in the baicalin-administered groups at doses of 25, 50 and 100 mg/kg (Fig. 5A). CASP-3 activation appears to be a prerequisite for apoptosis (He et al., 2013). Caspase-3 has virtually no activity until it is cleaved by an initiator caspase after apoptotic signaling events have occurred (Walters et al., 2009). These results indicate that CASP-3 cleavage was blocked by baicalin treatment. Inhibition of CASP-3 activation has anti-apoptotic effects in various systems (Huang et al., 2013). The results of the present study demonstrated that baicalin, which promoted BCL-2, inhibited BAX and blocked CASP-3 cleavage, had protective effects on mammary gland cells during S. aureus-induced mastitis. In the present study, we demonstrated the protective effects of baicalin on mammary gland cells in a mouse model of S. aureus-induced mastitis. The results indicated that baicalin treatment reduced TLR2 expression and p53 phosphorylation,
inhibiting apoptosis during S. aureus infection. Baicalin also displayed protective effects on mammary gland cells by regulating the expression of apoptosis-related factors (BCL-2, BAX and CASP-3) during S. aureus-induced mastitis. After further research, baicalin may be demonstrated to be effective in treating S. aureusinduced mastitis.
Acknowledgment This work supported by a grant from the National Natural Science Foundation of China (No.30972225, 30771596, 31130053) and Research Fund for the Doctoral Program of Higher Education of China (No. 20110061130010).
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Baicalin inhibits Staphylococcus aureus-induced apoptosis by regulating TLR2 and TLR2-related apoptotic factors in the mouse mammary glands Mengyao Guo, Yongguo Cao, Tiancheng Wang, Xiaojing Song, Zhicheng Liu, Ershun Zhou, Xuming Deng, Naisheng Zhang n, Zhengtao Yang n Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province 130062, People's Republic of China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 August 2013 Received in revised form 10 October 2013 Accepted 17 October 2013 Available online 7 November 2013
Baicalin, the major active constituent of the isolated root of Scutellaria baicalensis, is widely used in China and Southeast Asian countries. Evidence has indicated that baicalin has multiple biological activities, including anti-apoptotic properties. Mastitis is a severe problem in humans and other animals and is characterized by mammary gland cell apoptosis. Staphylococcus aureus (S. aureus) is the major pathogen that causes mastitis. This study was designed to evaluate the protective effects of baicalin on the mammary glands during S. aureus-induced mastitis. In the present study, a mouse model was infected with S. aureus to induce mammary gland injury. Baicalin treatment was administered between 6 and 24 h after infection. Toll-like receptor 2, p53, BAX, BCL-2 and caspase-3 expression were analyzed using qPCR and Western blotting. The results indicated that baicalin significantly attenuated pathological damage and cell death in the mammary glands. Further studies revealed that baicalin down-regulated the expression of Toll-like receptor 2 (TLR2) and the phosphorylation of p53 in the mammary glands after S. aureus-induced mastitis. Baicalin also promoted the expression of BCL-2 at the mRNA and protein levels but inhibited BAX and caspase-3 (CASP-3) cleavage. Baicalin inhibited apoptosis and had protective effects on mammary gland tissues during S. aureus-induced mastitis. These effects were displayed by reductions in TLR2 expression and p53 phosphorylation and the regulation of apoptosis-related factors (BCL-2, BAX and CASP-3) in mammary gland tissues. & 2013 Elsevier B.V. All rights reserved.
Keywords: Baicalin Staphylococcus aureus Mastitis Toll-like Receptor 2 Apoptosis
1. Introduction Mastitis, which is caused by infections of the mammary glands, poses a serious problem in humans and animals (Seegers et al., 2003). Mammary gland infections are caused by infectious bacterial pathogens (Carneiro et al., 2009). Gram-positive bacteria tend to cause mastitis, and present in the mammary glands for long periods of time, resulting in serious injury to mammary gland cells. The structure of breast tissue and lactation function are damaged, and the condition can sometimes even be fatal (Guo et al., 2013). Staphylococcus aureus is the major gram-positive pathogen, causing both community-acquired and nosocomial infections (Li et al., 2010a). To date, there is no efficacious treatment for S. aureus-induced mastitis. A mouse model of S. aureus-induced mastitis was first described by Chandler (1970). Anderson and Chandler further characterized this model in the mid-1980s (Anderson and Craven, 1984). Since 2000, S. aureus has n
Corresponding authors. Tel./fax: þ86 431 87835 140. E-mail addresses: [email protected] (N. Zhang), [email protected] (Z. Yang). 0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.10.032
been widely employed in studies using a mouse model of microbial mastitis. Thus, we chose to use this model in our study. Local innate immunity plays a key role in initiating and coordinating homeostatic and defense responses in the mammary glands. Once S. aureus infects the mammary glands, recognition of the infection through the activation of pattern recognition receptors (PRRs) is imperative for the initiation of the immune response in the mammary glands (Elazar et al., 2010). TLR2, a PRR receptor, is activated by peptidoglycan and lipoteichoic acid, which are major constituents of the cell wall pathogen-associated molecular pattern (PAMP) of gram-positive bacteria, including S. aureus (Whelehan et al., 2011). TLRs have been reported to increase in number and become activated in response to a bacterial challenge in the mammary glands (Petzl et al., 2008). Activation of TLR2 can trigger proapoptotic signals and cause cell death in various systems (Sabroe et al., 2005). Caspase activities were observed to increase significantly in TLR2 signaling activated cells (Li et al., 2009b). S. aureus has been reported to induce apoptosis in epithelial cells, lymphocytes, and macrophages (Kahl et al., 2000; Tucker et al., 2000), and TLR2 expression was observed to be upregulated after S. aureus infection (Zhao et al., 2008).
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2.2. Histological analyses and apoptosis detection
Fig. 1. Chemical structure of baicalin.
Baicalin (Fig. 1), the major active constituent of the isolated root of Scutellaria baicalensis, is widely used in China and Southeast Asian countries (Ma et al., 2009). Evidence has indicated that baicalin has multiple biological properties, including antiapoptotic (Cheng et al., 2012), anti-oxidant (Cao et al., 2011), anti-tumor, anti-ischemic, anti-inflammatory (Guo et al., 2013) and immune system modulatory activities (Li et al., 2010b). Baicalin has been reported to play a beneficial role in various experimental models of invasive toxicants by alleviating inflammatory injury (Kim and Lee, 2012) via the involvement of TLR2- or TLR4-mediated innate immune reactions (Hou et al., 2012; Li et al., 2012). In our previous study, baicalin was demonstrated to play an anti-inflammatory role in S. aureus-induced mastitis (Guo et al., 2013). The present study was conducted to determine the antiapoptotic effects of baicalin on the mammary glands in a mouse model of S. aureus-induced mastitis and to examine the mechanisms of action involved.
2. Materials and methods 2.1. Animal experimental groups and drug administration In total, 60 adult female BALB/c mice (6–8 weeks old, weighing 40–45 g) were used in the present study. The mice were provided by the Experimental Animal Center of Norman Bethune Medical College, Jilin University, China. All procedures were conducted according to the US NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Jilin University. Fifty milliliters of culture aliquots was centrifuged and washed with phosphate-buffered saline (PBS) prior to resuspension. S. aureus were resuspended in 1000 μl PBS (2 108 CFU per 30 μl). A 100 μl suspension was used per breast. The S. aureus suspension was injected via teat canal for inducing infection in the mammary gland. Baicalin was dissolved in physiological saline. Within the first 24 h after infection was induced in the mammary glands, baicalin was intraperitoneally injected four times (at 6, 12, 18 and 24 h). The mice were divided into three groups as follows (1) Microbionation group (MG): The mouse model of S. aureusinduced mastitis was established and the mice were treated with the vehicle control (saline). (2) Baicalin administration groups (BAGs): The mouse model of S. aureus-induced mastitis was established, and the mice were intraperitoneally administered baicalin at 25, 50, or 100 mg/kg (Guo et al., 2013). The dosage of baicalin was determined based on the results of a previous study. (3) Control groups (CGs): The mice were treated with normal saline (as a vehicle control) at the same volume and time points as baicalin treatment.
Mammary gland tissues were fixed in 10% formalin for two weeks and obtained from embedded paraffin samples. The tissues were deparaffinized with xylene and rehydrated using graded alcohol for the staining analyses. The sections were stained with hematoxylin and eosin (H&E) and then visualized using a microscope (Olympus, Japan). Apoptosis was identified in the mammary gland tissue sections using the in situ terminal deoxynucleotidyl transferase (TdT)mediated deoxyuridine triphosphate (dUTP) biotin nick-end labeling (TUNEL) technique with the In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany). Paraffin wax-embedded tissue sections were treated with proteinase K without DNase and incubated at 37 1C with the terminal TdT/ nucleotide mixture for 1 h. The reactions were then stopped, and the samples were rinsed with phosphate-buffered saline (PBS). Nuclear labeling was developed using horseradish peroxidase and diaminobenzidine. Hematoxylin was used for counterstaining. The evaluation of the apoptotic indices was performed by detecting positively stained nuclei at 400 magnification. 2.3. Quantitative real-time polymerase chain reaction Total RNA was isolated from the tissue samples (50 mg tissue; n¼ 3/ treatment group) using the TRIzol reagent according to the manufacturer's instructions (Invitrogen, China). The concentration and purity of the total RNA were determined spectrophotometrically at 260/280 nm. First-strand cDNA was synthesized from 5 μg of total RNA using oligo(dT) primers and Superscript II reverse transcriptase, according to the manufacturer's instructions (Invitrogen, USA). Synthesized cDNA was diluted five times with sterile water and stored at 80 1C. The Primer Premier software (PREMIER Biosoft International, USA) was used to design specific primers for TLR2, p53, BCL-2, BAX, CASP-3 and β-actin based on known sequences (Table 1). Quantitative real-time PCR was performed on an ABI PRISM 7500 Detection System (Applied Biosystems, USA). Reactions were performed in a 25-μl reaction mixture containing 12 μl of 2 SYBR Green I PCR Master Mix (TaKaRa, China), 2 μl of diluted cDNA, 0.5 μl of each primer (10 μM), 0.8 μl of 50 ROX reference Dye II, and 9.2 μl of PCR-grade water. The PCR procedure for TLR2, p53, BCL-2, BAX, Caspase-3 and β-actin consisted of 95 1C for 30 s, followed by 35 cycles of 95 1C for 15 s, 63 1C for 30 s and 60 1C for 30 s. A melting curve analysis showed only one peak for each PCR product. A dissociation curve was run for each plate to confirm the production of a single product. The amplification efficiency for each gene was determined using the DART-PCR program (Peirson et al., 2003). The mRNA relative abundance was calculated according to the method of Pfaffl (2001), accounting for genespecific efficiencies and was normalized to the mean expression of β-actin. Results (fold changes) were expressed as 2-ΔΔCt in which ΔΔCt ¼ (CtTLR2/p53/BCL-2/BAX//CASP-3-Ctβ-actin)t (Ct TLR2/p53/ BCL-2/BAX//CASP-3 Ct β-actin) c, where Ct TLR2/p53/BCL-2/BAX/CASP-3 and Ctβ-actin are the cycle thresholds for TLR2/p53/BCL-2/BAX/CASP-3 and β-actin genes in the differently treated groups, respectively, t is the treatment group, and c is the control group. 2.4. Western blot analyses Mammary gland tissues were weighed and homogenized with phosphate-buffered saline (w/v: 1/9) on ice, and the total protein was then extracted according to the manufacturer's recommended protocol (Invitrogen, Beijing, China). The protein concentrations were determined using the BCA Protein Assay Kit. Samples with equal amounts of protein (50 μg) were fractionated on 10% SDS
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Table 1 Oligonucleotide primers used for qPCR. Name
TLR2 p53 BAX BCL-2 CASP-3 β-Actin
Primer sequence
Product size (bp)
Sense: 5'- TGTGCCACCATTTCCACG-3' Anti-sense: 5'-AAAGGGCGGGTCAGAGTT-3' Sense: 5′-GGGAATAGGTTGATAGTTGTC-3' Anti-sense: 5′-CAGGCTTTGCAGAATGGA-3' Sense: 5′-AGTCCTGCGGGGCGGAGGCCATGTT-3' Anti-sense: 5′-AACATGGCCTCCGCCCCGCAGGACT-3' Sense: 5′-CTACCGTCGTGACTTCGC-3' Anti-sense: 5′-GGGTGACATCTCCCTGTT-3' Sense: 5′-AATCTGACGGTCCTCCTG-3' Anti-sense: 5′-TCGCCAAATCTTGCTAAT-3' Sense: 5′-TAAAACGCAGCTCAGTAACAGTCGG-3' Anti-sense: 5′–TGCAATCCTGTGGCATCCATGAAAC-3'
188 181 194 184 198 182
Fig. 2. Histopathology of mammary tissue after infecting with S. aureus ( H&E staining 100 ). (A) Mammary tissues of CG. (B) Microbionation group (MG). ((C)–(E)) Baicalin groups, administered at 25, 50, or 100 mg/kg.
polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and blocked in 5% skim milk in PBST for 1.5 h at 25 71 1C. The membranes were then incubated at 4 1C overnight with 1:1000 dilutions (v/v) of the primary antibodies. The primary antibodies were including TLR2 (cat number GTX13855, GeneTex, Inc. USA, clone 24088), p53 (cat number 2524s Cell Signaling Technology, Inc. USA, clone 1C12 ), p-p53(cat number 9284s Cell Signaling Technology, Inc. USA, clone Ser15 ), BCL-2 (cat number 611902, Biolegend Inc. San Diego, USA, clone Poly6119), BAX(cat number 625102, Biolegend Inc. USA, clone Poly6251), CASP-3 (cat number sc-136219, Santa Cruz Biotechnology Inc. USA, clone 8B1.1), cleaved CASP-3 (cat number GTX86951, GeneTex, Inc., USA, clone Asp175) and β-actin (cat number KM9001, Sungene Biotech Co., Ltd, China, clone 6G3). After washing the membranes with PBST, incubations with 1:5000 dilutions (v/v) of the secondary antibodies were conducted for 1.5 h at 25 71 1C. The secondary antibodies were HRP-conjugated goat–mouse antibodies (cat number LK2003L, Sungene Biotech Co., Ltd, China). (name, supplier, cat number, clone, etc.) Protein expression was detected using an Enhanced Chemiluminescence Detection System. β-Actin was used as a loading control.
2.5. Data analyses Statistical analyses were performed using the SPSS software package (ver. 13 for Windows; SPSS Inc., Chicago, IL, USA). Significance was determined using a one-way ANOVA with a significance level of P o0.05. The data were assessed using the Tukey–Kramer method for multiple comparisons. All values are expressed as the means 7 S.D.
3. Results 3.1. Histopathological changes Mammary gland tissues were harvested 24 h after inducing infection and baicalin treatment. Pathological changes were not observed in the CG (Fig. 2A). In the MG (Fig. 2B), the lobules of the mammary glands were incomplete (mammary gland alveolar congestion and marked thickening of the alveolar walls were observed, the acini of the mammary glands were damaged, and the epithelial cells were destroyed). Many inflammatory cells,
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including neutrophils and macrophages, were observed in the mammary gland tissues. However, these histopathological changes were ameliorated in the BAG following treatment with baicalin doses of 25, 50 and 100 mg/kg (Fig. 2C–E). Although some inflammatory cells were observed among the mammary gland tissues, the lobules were complete and the acini were not destroyed.
3.2. Apoptosis detection Apoptotic mammary gland cells were detected using the TUNEL assay. Apoptosis was not observed in the CG (Fig. 3A). Apoptosis was significantly increased in the MG (Fig. 3B) compared to the CG and BAGs. The majority of the apoptotic mammary gland cells were found in the breast tissues that exhibited structural failures. Baicalin significantly reduced apoptosis in the BAGs compared to the MG, and apoptosis was ameliorated in the BAG following treatment with doses of 25, 50 and 100 mg/kg (Fig. 3C–E).
3.4. Baicalin suppressed p53 expression in the mammary glands during S. aureus-induced mastitis p53 is a key signaling molecule during the development of apoptosis. Ordinarily, p53 is phosphorylated to promote apoptosis. The mRNA expression levels of p53 were measured using quantitative RT-PCR, and the protein expression levels of p53 and p53 phosphorylation were measured using Western blot analyses (Fig. 5). p53 mRNA levels were significantly increased (P o0.05) in the MG and baicalin-administered groups compared to the CG. However, the p53 mRNA levels of the MG group did not differ from those in the baicalin-administered groups (at doses of 25, 50, or 100 mg/kg) in a statistically significantly manner (P 40.1). The protein levels of phosphorylated p53 were significantly increased in the MG and were suppressed by baicalin treatment (Fig. 5A). Phosphorylated p53 protein levels were decreased in the baicalinadministered groups compared to the MG. The protein expression levels of unphosphorylated p53 protein did not differ between the CG, MG and baicalin-administered groups. 3.5. Effects of baicalin on BAX, BCL-2 and CASP-3 expression in the mammary glands during S. aureus-induced mastitis
3.3. Effects of baicalin on TLR2 expression in the mammary glands To determine the effects of baicalin on TLR2 expression in S. aureus-induced mastitis, the mRNA and protein expression levels of TLR2 were measured using quantitative RT-PCR and Western blot analyses. The results are shown in Fig. 4. Stimulation with S. aureus led to robust increases in TLR2 mRNA and protein expression levels. These effects were significantly inhibited by baicalin treatment. The mRNA and protein expression levels were significantly increased (P o0.05) in the MG and were slightly elevated in the baicalin-administered groups compared to the levels in the CG. The levels were significantly reduced in the baicalin-administered groups compared to the levels in the MG. As the dose of baicalin increased, the effects tended to become more obvious. The results are shown in Fig. 4.
CASP-3 activation is well-established to play a crucial role in the apoptotic response. BAX and BCL-2 play common roles in regulating CASP-3 activation or suppression. We investigated whether BAX, BCL-2 and CASP-3 were affected by baicalin treatment in S. aureus-induced mastitis. The results are shown in Fig. 5. BAX expression was significantly increased at both the mRNA and protein levels in the MG (Fig. 6). BAX mRNA levels were reduced in the baicalin-administered groups at doses of 25, 50 and 100 mg/kg, and BAX protein levels were also significantly reduced in the baicalin-administered groups (Fig. 6A). BCL-2 protein and mRNA expression were significantly reduced in the MG compared to the CG, but BCL-2 expression was significantly increased in the baicalinadministered groups (Fig. 6). CASP-3 mRNA levels were significantly increased in the MG and BAGs (Fig. 6). The mRNA levels of CASP-3 did not differ from those in the MG in the baicalin-administered
Fig. 3. Apoptosis detection of mammary tissue after S. aureus infection (TUNEL staining 100 ). (A) Mammary gland tissues in the CG. (B) Mammary gland tissues in the microbionation group (MG). ((C)–(E)) Mammary gland tissues in the baicalin-treated groups (25, 50, or 100 mg/kg). The nucleus of apoptotic cells were indicated brown staining, showed by arrows.
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Fig. 4. Effects of baicalin on TLR2 in the mammary glands. (A) TLR2 protein levels in mammary tissue. (B) TLR2 mRNA levels in mammary tissue. Western blotting was performed to detect TLR2 protein levels in the control group (CG), the microbionation group (MG), and the baicalin administration groups (25, 50, or 100 mg/kg). qPCR was performed to detect the mRNA levels of TLR2. β-Actin was used as a control. The values are presented as the means 7 S.E.M. (n¼ 10). nPo 0.01 indicates a significant difference from the CG. # Po 0.01 indicates a significant difference from the MG.
groups at doses of 25, 50 and 100 mg/kg. CASP-3 protein expression was also significantly reduced, but cleaved CASP-3 protein levels were significantly increased in the MG. CASP-3 protein levels were not reduced in the baicalin-administered groups at doses of 25, 50 and 100 mg/kg, but cleaved CASP-3 protein levels were significantly reduced (Fig. 6A). As the dose of baicalin increased, the effects on BAX, BCL-2 and CASP-3 expression tended to become more obvious.
4. Discussion Mastitis, which is mainly caused by microbial infections, is a severe problem in humans and other animals (Seegers et al., 2003). There are two types of mastitis: clinical and subclinical (Kim et al., 2011). The mouse model of S. aureus-induced mastitis is the current standard (Notebaert and Meyer, 2006). In our previous study, we were able to successfully establish this model (Guo et al., 2013), and this model was also used in the present study. Histopathological changes were observed in the present study. In the MG, the lobules of the mammary glands were damaged and incomplete (the alveolar glands were congested, marked thickening of the alveolar walls was observed, and the epithelial cells were destroyed) (Fig. 2B). These observations were likely due to
S. aureus affecting the mammary tissue epithelial and endothelial cells, which is consistent with the results of previous research (Anaya-Lopez et al., 2006). Some inflammatory cells, including neutrophils and macrophages, were observed in the mammary gland tissues in the MG. These results were similar to those of our previous study (Guo et al., 2013). Apoptosis was significant in the MG (Fig. 3B). Most of the apoptotic mammary gland cells were found in the breast tissues that exhibited structural failures. Baicalin protected mammary tissue epithelial and endothelial cells and reduced mammary damage (Fig. 2C–E). Mammary gland injury was ameliorated in the baicalin-administered groups. Apoptosis was significantly ameliorated in the BAGs following the administration of doses of 25, 50 and 100 mg/kg (Fig. 3C–E). These results indicated that mammary gland tissues that had been infected by S. aureus were protected by baicalin treatment. In previous studies, TLR2, a key immune receptor that is a member of the TLR family, was observed to be activated by S. aureus infection in the mammary glands, and TLR2 expression was increased following infection (Yang et al., 2008). In the present study, TLR2 mRNA and protein levels were assessed and were found to be significantly increased in the MG compared to the baicalin-administered groups. The results of the present study were similar to those of the previous study. The S. aureus cell wall components peptidoglycan and lipoteichoic acid have been found
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Fig. 5. Effects of baicalin on p53 expression. (A) p53 protein levels in mammary tissue. (B) p53 mRNA levels in mammary tissue. Western blotting was performed to detect p53 protein levels and p53 phosphorylation in the control group (CG), the microbionation group (MG), and the baicalin administration groups (25, 50, or 100 mg/kg). qPCR was performed to detect the mRNA levels of p53. β-Actin was used as a control. The values are presented as the means 7S.E.M. (n¼ 10). nPo 0.01 indicates a significant difference from the CG. # Po 0.01 indicates a significant difference from the MG.
to activate TLR2 (Li et al., 2009a). Activation of TLR2 signaling mediates immune cell activation and cell death in various systems (Sabroe et al., 2005). In the present study, TLR2 mRNA and protein levels were reduced in the baicalin-administered groups. TLR2 overexpression has been reported to result in cellular susceptibility to apoptosis (Fan et al., 2005). Thus, our findings indicated that baicalin treatment had an anti-apoptotic effect, reducing the expression of TLR2 in the mammary glands following S. aureus infection. p53 is defined as a tumor suppressor and nuclear transcription factor and can regulate several major cellular functions, including the cell cycle, senescence, and cell death (Culmsee and Mattson, 2005). Previous studies have reported that p53 may mediate apoptosis that is induced by a range of insults, including DNA damage, hypoxia, withdrawal of trophic support, hypoglycemia, oxidative stress, and viral infections (Morrison et al., 2003). In the present study, p53 mRNA levels were significantly increased (Po0.05) in the MG and baicalin-administered groups. These results indicated that p53 could be activated by S. aureus infections in the mammary glands. p53 mRNA levels in the baicalin-administered groups did not significantly differ (P40.1) from those in the MG. However, the protein levels of phosphorylated p53 were suppressed by baicalin treatment (Fig. 4A). Phosphorylated p53 protein levels were decreased in the baicalinadministered groups compared to the MG. This result indicated that
baicalin had anti-apoptotic effects in mammary gland cells by suppressing p53 phosphorylation. Activation of TLR2 also triggers the activation of proapoptotic signals, including caspase activity (Yin et al., 1997). The results of the previous study indicated that CASP-3 activity increased significantly in TLR2 signaling activated cells (Fan et al., 2005). p53 plays roles in both the intrinsic and extrinsic apoptotic pathways. Activation of p53 results in the expression of proapoptotic members of the Bcl-2 family (Miyashita and Reed, 1995; Oda et al., 2000) and death receptors (Muller et al., 1998). The results of the previous study revealed that the proapoptotic protein Bax was activated by p53 phosphorylation and binding to the antiapoptotic protein Bcl-2, which resulted in the induction of apoptosis (Chipuk et al., 2004). To further characterize the nature of the protective effects of baicalin against apoptosis, we examined the effects of baicalin on BAX, BCL-2 and CASP-3 expression. Ordinarily, BCL-2 mediated the activation and translocation of BAX to the mitochondrial outer membrane (Chipuk and Green, 2006). BAX and BCL-2, which act as death agonists and antagonists, respectively, are two opposing proteins (Kroemer, 1997). Apoptosis occurs as a result of competing dimerization between the two proteins, the relative proportion of which ultimately controls the sensitivity or resistance of cells to apoptotic stimuli (Reed, 1994). Our results indicated that BAX mRNA and protein
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Fig. 6. Effects of baicalin on the activation of apoptosis-related factors. (A) BCL-2, BAX, and CASP-3 protein levels in mammary tissue. (B) BCL-2, BAX, and CASP-3 mRNA levels in mammary tissue. Western blotting or qPCR was performed to detect BCL-2, BAX, and CASP-3 protein or mRNA levels in the control group (CG), the microbionation group (MG), and the baicalin administration groups (25, 50, or 100 mg/kg). β-Actin was used as a control. The values are presented as the means 7S.E.M. (n ¼10). nPo 0.01 indicates a significant difference from the CG. # P o0.01 indicates a significant difference from the MG.
expression were increased in the MG and were reduced in the baicalin-administered groups. However, BCL-2 mRNA and protein levels were significantly increased (P o0.05) in the baicalinadministered groups compared to those in the MG. The relative proportion (BAX/BCL-2) was also higher in the MG than in the baicalin-administered groups. This result indicated that S. aureus induced apoptosis in the mammary glands, which was suppressed by baicalin treatment, and was consistent with the histopathological changes that were observed. CASP-3 is a major proapoptotic element. TLR2 can trigger CASP-3 activation and cause cell death and apoptosis (Harburg et al., 2007). BAX and BCL-2 also play common roles in regulating CASP-3 activation or suppression (Shi et al., 2010). In the present study, we observed striking effects on CASP-3 mRNA or protein expression levels following baicalin treatment. CASP-3 mRNA levels and cleaved CASP-3 bands (19 Kd and 17 Kd bands) were significantly increased in the MG (Fig. 5). The mRNA levels of CASP-3 were not influenced by baicalin treatment. The cleaved CASP-3 proteins were significantly reduced in the baicalin-administered groups at doses of 25, 50 and 100 mg/kg (Fig. 5A). CASP-3 activation appears to be a prerequisite for apoptosis (He et al., 2013). Caspase-3 has virtually no activity until it is cleaved by an initiator caspase after apoptotic signaling events have occurred (Walters et al., 2009). These results indicate that CASP-3 cleavage was blocked by baicalin treatment. Inhibition of CASP-3 activation has anti-apoptotic effects in various systems (Huang et al., 2013). The results of the present study demonstrated that baicalin, which promoted BCL-2, inhibited BAX and blocked CASP-3 cleavage, had protective effects on mammary gland cells during S. aureus-induced mastitis. In the present study, we demonstrated the protective effects of baicalin on mammary gland cells in a mouse model of S. aureus-induced mastitis. The results indicated that baicalin treatment reduced TLR2 expression and p53 phosphorylation,
inhibiting apoptosis during S. aureus infection. Baicalin also displayed protective effects on mammary gland cells by regulating the expression of apoptosis-related factors (BCL-2, BAX and CASP-3) during S. aureus-induced mastitis. After further research, baicalin may be demonstrated to be effective in treating S. aureusinduced mastitis.
Acknowledgment This work supported by a grant from the National Natural Science Foundation of China (No.30972225, 30771596, 31130053) and Research Fund for the Doctoral Program of Higher Education of China (No. 20110061130010).
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