Subacute ruminal acidosis (SARA) is a nutrition-associated metabolic disease with high incidence rate in dairy cows. Studies in the United States have indicated that up to 19% of early-lactation dairy cows and 26% of mid-lactation cows have SARA (Enemark, Reference Enemark2008). Highly fermentable diets are often fed to high producing lactating cows to increase milk production, which can reduce ruminal pH, microbial activity and increase the risk of SARA (Pan et al., Reference Pan, Yang, Xue, Xin, Jiang, Xiong and Beckers2016). Dairy cows with SARA display ruminal inflammation (rumenitis), which is associated with high a concentration of lipopolysaccharide (LPS) in the rumen (Gozho et al., Reference Gozho, Krause and Plaizier2007; Dionissopoulos, Reference Dionissopoulos, Laarman, AlZahal, Greenwood, Steele, Plaizier, Matthews and McBride2013). LPS interacts with a class of receptors known as toll-like receptor 4 (TLR4) and activates various inflammatory pathways mediated by mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF-κB) (Nyati et al., Reference Nyati, Masuda, Zaman, Dubey, Millrine, Chalise, Higa, Li, Standley and Saito2017), which promote the expression of pro-inflammatory mediators including the cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) (Di et al., Reference Di, Huang and Ho2011; Chen et al., Reference Chen, Chen, Pan, Li, Tu, Liu, Dai, Zhang, Sun and Feng2015). Wellnitz and Kerr (Reference Wellnitz and Kerr2004) have reported that LPS interacts with epithelial cells and stimulates the production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, which is further involved in the induction of inflammation (Wellnitz and Kerr, Reference Wellnitz and Kerr2004). Zhang et al. (Reference Zhang, Zhu and Mao2016) also reported that LPS stimulation can induce high levels of mRNA expression of various cytokines in the rumen epithelial cells of Holstein cows, especially the IL-1β. Therefore, the rumenitis may be a direct result of high level of ruminal LPS induced by SARA.
Berberine (BBR) is the main component of the Chinese medicine Rhizoma Coptidis, which is often used to treat inflammation of the gastrointestinal tract in human (Chen et al., Reference Chen, Yu, Li, Fichna and Ston2014). It has been reported that BBR suppresses the expression of IL-1β in LPS-treated THP-1 cells (Zhou et al., Reference Zhou, Feng, Xu, Sun, Ma, Zhang, Liu, Xu, Wu and Shen2017). Furthermore, Guo et al. (Reference Guo, Shih-Lung, Guo, Li, Zheng, Rachel, Liu, Pei, Xu and Cai2016) also proved that BBR ameliorated liver inflammation and decreased hepatic IL-1β and TNF-α mRNA levels (Guo et al., Reference Guo, Shih-Lung, Guo, Li, Zheng, Rachel, Liu, Pei, Xu and Cai2016). Berberine is widely used in veterinary clinics in China. Zhou et al. (Reference Zhou, Yang, Li, Liu and Wang2015) have reported that berberine could prevent and treat multi-drug resistant S. epidermidis mastitis in dairy cows (Zhou et al., Reference Zhou, Yang, Li, Liu and Wang2015). However, whilst an anti-inflammatory role for BBR is generally accepted, little detail of the mechanism has been revealed, and BBR has not previously been studied in LPS-stimulated bovine ruminal epithelial cells (REC). Our hypothesis was that BBR would have an anti-inflammatory effect on LPS-stimulated REC, and in addition we wished to understand the underlying mechanism. The results of this study will lay a foundation for the possible application of BBR on the prevention and treatment of rumenitis induced by SARA.
Material and methods
The Ethics Committee on the Use and Care of Animals at Jilin University approved the study protocol (Changchun, China).All animals use in this study were approved by the Chinese Society of Laboratory Animal Sciences (2015 clinical trial [2015-121]).
Rumen epithelial cell culture
The method of Sun et al. (Reference Sun, Yuan, Chen, Wang, Wang, Sun, Li, Li and Liu2017) was used to isolate and prepare rumen epithelial cells for culture. Briefly, ruminal epithelia were collected under terminal anaesthesia from newborn female Holstein calves weighing 35–45 kg. Washed epithelia were minced and digested with trypsin. After centrifugation, cell suspensions were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum (FBS) (HyClone, Logan, UT) and filtered with 300 mesh (37.5 µm) cell sieves. The trypan blue dye exclusion method was used to assess cell viability. The cell density was adjusted to 5 × 104, 1 × 105, or 1 × 106 cells/ml. The cell suspensions were seeded sequentially into 96-well tissue culture plates (100 µl/well) for CCK8 assay. The cell suspensions seeded into 24-well plates (and covered by glass coverslips) was 1 ml/well, while 6-well tissue culture plates received 2 ml/well and were incubated at 37 °C in 5% CO2. The medium composed of DMEM containing 15% FBS, 6 µg/ml gentamicin, 6 µg/ml amphotericin B, 200 mg/ml streptomycin and 200 U/ml penicillin was replaced every 24 h. Full details of the cell preparation and culture are given in the online Supplementary File.
CCK-8 assay
Cell counting kit-8 (CCK-8) is used for simple and accurate cell proliferation and toxicity analysis. REC were added to 96-well culture plates and adjusted to 1 × 105 cells/ml. There were 100 µl cells in each well which were cultured for 12 h. The cell-free supernatants were discarded. After incubation with BBR (10, 25, 50, 70, or 100 µm, 50 µl/well; Sigma-Aldrich, MO, USA) for 1 h, the cells were stimulated by LPS (4 µg/ml, 50 µl/well; Sigma-Aldrich, MO, USA) for 18 h. CCK8 was added into all of the experimental groups (10 µl/well; Sigma-Aldrich, MO, USA) and incubated for 3 h. The OD value was measured at 450 nm using a microplate reader (Thermo Scientific Instrument Inc., Shanghai, China).
Enzyme-linked immunosorbent assay (ELISA)
REC were treated with different concentrations of BBR (50, 70, or 100 µm) and 4 µg/ml LPS in 24-well plates (1 × 106 cells/ml). Cell-free supernatants were subsequently centrifuged at 3000 rpm for 20 min to extract the liquid in the upper layer for using in assays for the inflammatory cytokines TNF-α, IL-1β, and IL-6 by ELISA kits (TNF-α: ml024586, 6.3–8000 pg/ml; IL-6: ml023756, 1–200 pg/ml; IL-1β: ml023753, 1–640 pg/ml; Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's instructions, respectively.
RNA extraction and real-time PCR
Standard methods were used to isolate total RNA for reverse-transcription into cDNA. The mRNA expression levels were evaluated by quantitative polymerase chain reaction (qRT-PCR) analysis using SYBR Green and gene expression levels were analyzed with the 2 − ΔΔCT method, where CT is the cycle threshold (Rao et al., Reference Rao, Huang, Zhou and Lin2013). Details are given in the online Supplementary File, including the primers used (online Supplementary Table S1). Primers were designed using Primer 5.0 (Canada Premier Company), the gel electrophoresis data are in Supplementary Fig. S3 and the melt curve analysis is shown at Supplementary Fig. S4.
Western blotting
Standard Western blotting techniques were used and full details are given in the online Supplementary File. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation was performed with 40 µg of protein per lane and with known molecular weight markers (Sangon Biotech Co., Ltd.). Target proteins were transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% nonfat dry milk. The blocked membranes were hybridized overnight at 4 °C with antibodies against NF-κB p-p65, TLR4, MyD88 (ab86299, ab22048, ab2068, Abcam, Cambridge, MA, USA), NF-κB p65, IκBα, p-IκBα, JNK, p-JNK, ERK, p-ERK, p38MAPK, p-p38MAPK (4764, 4814, 9246, 9252, 9251, 4695, 4370, 8690, 4511, Cell Signaling Technology, Danvers, MA, USA) and β-actin (sc-47778,Santa Cruz, CA, USA) respectively. Then the membranes were washed and incubated with peroxidase-conjugated secondary antibody (1:5000 dilutions in TBST) at room temperature for 45 min. The final blots were developed using enhanced chemiluminescence solution (Pierce Biotechnology Inc., Chicago, IL, USA) in a Western blotting detection system (ProteinSimple, Santa Clara, CA, USA).
Immunocytofluorescence
Cells were cultured on glass coverslips at a density of 5 × 104 cells/well in a 24-well culture plate. They were grown to approximately 90% confluency and treated with 100 µm BBR and 4 µg/ml LPS. The coverslips were then washed three times with PBS, fixed with a 4% paraformaldehyde/PBS for 20 min at room temperature, washed three times with PBS, and then subjected to antigen retrieval with EDTA-Na2 (95 °C, 5 min), after which they were rewashed. Following treatment with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) and washing with PBS three times, the cells were blocked with a goat serum-containing blocking buffer. After further washes, the slides were incubated with primary antibody against NF-κB p65 overnight at 4 °C and then exposed to goat anti-rabbit IgG conjugated with Cy3 (Beyotime Biotechnology Inc.) for 30 min. and counterstained with Hoechst 33258 (Beyotime Biotechnology Inc.). Each step included three washes with PBS. Immunofluorescence microscopy was performed using a confocal laser microscope (FluoView FV1200, Olympus, Tokyo, Japan).
Statistical analysis
All data were analyzed with SPSS (Statistical Package for the Social Sciences) 16.0 software (SPSS Incorporated, Chicago, IL, USA). The data with more than two groups were tested with ANOVA with subsequent Bonferroni correction and expressed as the mean ± standard error of mean (sem). P < 0.05 was considered to be statistically significant.
Results
Effects of BBR on cell viability
The potential cytotoxicity of BBR (in absence or presence of LPS) on REC was analyzed by CCK-8 assay. Cell viabilities were not affected by BBR (10, 25, 50, 70, and 100 µm) and LPS (4 µg/ml) (online Supplementary Fig. S1). The morphology of treated and non-treated REC did not differ (Supplementary Fig. S2).
Effects of BBR on the Expression of Inflammatory Cytokines
The levels of pro-inflammatory cytokines were measured using ELISA kits. LPS stimulated the expected increase in the contents of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 (Fig. 1A, C and E). Importantly, BBR treatment significantly decreased the concentrations of TNF-α, IL-1β, and IL-6 in LPS-treated REC in a dose-dependent manner (Fig. 1A, C and E). To further investigate the effect of BBR on the pro-inflammatory cytokines, the mRNA expression levels of the cytokines were measured by qRT-PCR. The results showed that BBR treatment significantly decreased the mRNA expression of TNF-α, IL-6, and IL-1β in LPS-stimulated REC (Fig. 1B, D and F). Overall, these results indicate that BBR can significantly decrease the synthesis of pro-inflammatory factors (TNF-α, IL-1β, and IL-6) in LPS-treated REC.
Fig. 1. Effects of different concentrations of BBR on the production of inflammatory mediators. REC was treated with different concentrations of BBR (50, 70, or 100 µm) and 4 µg/ml LPS in 24-well plates (1 × 106 cells/ml). The levels of TNF-α, IL-1β and IL-6 in the LPS-induced inflammation in the rumen epithelial cells were measured by ELISA. (A) The concentration of TNF-α. (B) The mRNA level of TNF-α. (C) The concentration of IL-1β. (D) The mRNA level of IL-1β. (E) The concentration of IL-6. (F) The mRNA level of IL-6. Similar results were obtained from three independent experiments. The data are presented as the mean ± sem (n = 6 in each group). a, b, c, and d: the same letter indicates the lack of significant difference (P > 0.05), whereas different letters indicate significant difference (P < 0.05).
BBR suppresses TLR4-mediated NF-κB and MAPK pathways
TLR4 and MyD88 are upstream molecular targets of NF-κB and MAPK and can directly regulate the activation of these pathways in LPS-induced inflammation (Nyati et al., Reference Nyati, Masuda, Zaman, Dubey, Millrine, Chalise, Higa, Li, Standley and Saito2017). Our results showed that BBR down-regulated the expression of TLR4 and MyD88 in LPS-treated REC in a dose-dependent manner (Fig. 2).
Fig. 2. Effects of BBR on the protein expression of TLR4 and MyD88. REC was treated with different concentrations of BBR (50, 70, or 100 µm) and 4 µg/ml LPS in 6-well plates (1 × 106 cells/ml). Protein samples were analyzed by Western blotting with specific antibodies; β-actin was used as a control. (A) The Western blotting results for TLR4 and MyD88. (B) The protein level of TLR4. (C) The protein level of MyD88. Similar results were obtained from three independent experiments. The data are presented as the mean ± sem (n = 6 in each group). a, b, c, and d: the same letter indicates the lack of significant difference (P > 0.05), whereas different letters indicate significant difference (P < 0.05).
The NF-κB and MAPK pathways play important roles in the induction of inflammatory cytokines. We, therefore, investigated the effects of BBR on the NF-κB and MAPK pathways in LPS-treated REC. As shown in Figs. 3 and 4, LPS treatment over-activated the NF-κB and MAPK inflammatory pathways. The phosphorylation levels of p65 and IκBα in the NF-κB pathway and the phosphorylation levels of p38MAPK, ERK, and JNK in the MAPK pathway were markedly increased. In contrast, BBR (50, 70, and 100 µm) inhibited the phosphorylation of IκBα and p65, which indicated that BBR treatment inhibited the over-activation of the NF-κB signaling pathway induced by LPS in REC. Furthermore, BBR treatment could significantly inhibit the phosphorylation of JNK and p38MAPK. However, there was no significant effect on the phosphorylation of ERK. These results indicate that BBR treatment can significantly decrease the activation of NF-κB- and MAPK-mediated inflammatory pathways in LPS-treated REC.
Fig. 3. Effects of BBR on NF-κB activation. REC was treated as described in Fig. 2. Protein samples were analyzed by Western blotting with specific antibodies; β-actin was used as a control; p indicates phosphorylation. Similar results were obtained from three independent experiments. The data are presented as the mean ± sem (n = 6 in each group). a, b, c, and d: the same letter indicates the lack of significant difference (P > 0.05), whereas different letters indicate significant difference (P < 0.05).
Fig. 4. Effects of BBR on the MAPK pathway. REC was treated as described in Fig. 2. Protein samples were analyzed by Western blotting with specific antibodies; β-actin was used as a control; p indicates phosphorylation. (A) The Western blotting results for JNK, p-JNK, ERK, p-ERK, p38MAPK, and p-p38MAPK. (B) The protein level of p-JNK. (C) The protein level of p-ERK. (D) The protein level of p-p38MAPK. Similar results were obtained from three independent experiments. The data are presented as the mean ± sem (n = 6 in each group). a, b, c, and d: the same letter indicates the lack of significant difference (P > 0.05), whereas different letters indicate significant difference (P < 0.05).
To further investigate the effect of BBR on the NF-κB pathway, the nuclear localization of NF-κB p65 was detected by immunocytofluorescence. NF-κB p65 was translocated to the nucleus of REC after LPS treatment, whereas BBR (100 µm) treatment partially prevented this phenomenon (Fig. 5), which further demonstrated that BBR attenuated the activation of LPS-induced NF-κB inflammatory pathway in REC.
Fig. 5. Effects of BBR on the transcriptional activity and immunofluorescence staining of NF-κB p65 in REC. REC was treated with 4 µg/ml LPS and 100 µm BBR. Cellular localization of p65 was analyzed by immunofluorescence using laser confocal microscopy (400×).
Discussion
SARA is a common disease in high-yielding dairy cows that are fed highly digestible diets. A reduction in ruminal pH below 5.6 for more than 3 h per day is characteristic of SARA (Gozho et al., Reference Gozho, Plaizier, Krause, Kennedy and Wittenberg2005). During low ruminal pH, gram-negative bacteria are lysed more rapidly, increasing the concentration of LPS in the rumen (Khafipour et al., Reference Khafipour, Plaizier, Aikman and Krause2011). It has been reported that intramammary infections caused by E. coli are more easily identified because they are characterized by a severe inflammatory response, including sudden shock, sepsis, and often death, caused by the releasing of LPS (Moretti et al., Reference Moretti, Biffani, Tiezzi, Maltecca, Chessa and Bozzi2017). The ruminal epithelium is continuously exposed to high level of LPS in cows with SARA. LPS is an inflammatory inducer and is involved in the development of rumenitis. Zhang et al. (Reference Zhang, Zhu and Mao2016) also reported that LPS is involved in the development of inflammation in cows with SARA (Zhang et al., Reference Zhang, Zhu and Mao2016). BBR has been reported to have an anti-inflammatory effect (Jeong et al., Reference Jeong, Hsu, Lee, Ham, Huh, Shin, Kim and Kim2009; Zha et al., Reference Zha, Liang, Xiao, Studer, Hylemon, Pandak, Wang, Li and Zhou2010). In the present study, we investigated the anti-inflammatory effects and mechanism of BBR on LPS-stimulated REC. Our results showed that BBR suppressed the expression of pro-inflammatory cytokines by preventing TLR4-mediated NF-κB and MAPK activation in LPS-stimulated REC, indicating that the anti-inflammatory mechanism of BBR was associated with suppressing TLR4-mediated NF-κB and MAPK signaling pathways.
Rumenitis is the direct result of SARA in dairy cows. Janssens and Beyaert (Reference Janssens and Beyaert2003) reported that LPS could cause inflammatory damage of ruminal epithelial cells in dairy cows with forestomach atony (Janssens and Beyaert, Reference Janssens and Beyaert2003). Importantly, Zhang et al. (Reference Zhang, Zhu and Mao2016) found that LPS treatment could significantly increase the mRNA expression of IL-1β and IL-8 in ruminal epithelial cells of Holstein cows in vitro (Zhang et al., Reference Zhang, Zhu and Mao2016). These studies indicated that the ruminal epithelial cells are capable of responding to the release of inflammatory cytokines. Thus, we used ruminal epithelial cells to investigate the anti-inflammatory mechanisms of BBR in vitro. Our results showed that LPS significantly increased the expression and production of inflammatory cytokines TNF-α, IL-6, and IL-1β in ruminal epithelial cells. These further mediated the damage caused by inflammation in the ruminal epithelium. Interestingly, BBR treatment significantly decreased the overproduction of inflammatory cytokines induced by LPS in a dose-dependent manner, which indicated that BBR could improve LPS-induced inflammation in the ruminal epithelium.
TLR4, one of the best characterized TLRs, is a pattern recognition receptor for LPS (Li et al., Reference Li, Zhang, Dong, Zhang, Sun and Qian2016; Wang et al., Reference Wang, Zhang, He, Yang, Wang, Shan, Li, Sun and Wu2018). TLR4 activates MyD88 and then induces the activation of NF-κB and MAPK signaling pathways to increase the release of pro-inflammatory cytokines (Lu et al., Reference Lu, Yeh and Ohashi2008). The increasing LPS could activate TLR4 on the surface of mammary epithelial cells, resulting in up-regulated DNA binding activity of nuclear factor (NF)-κB and increased expression of inflammatory cytokines. It would act synergistically to disrupt mammary epithelial function, and cause inflammation in the whole body (Wang et al., Reference Wang, Zhang, He, Yang, Wang, Shan, Li, Sun and Wu2018). In this study, Western blotting results showed that BBR attenuated LPS-induced TLR4 and MyD88 expression in REC, which indicated that BBR could inhibit the LPS-induced TLR4 pathway. NF-κB p65 is a nuclear transcription factor that exists in an inactive form in the cytoplasm bound to its inhibitor IκB (Shi et al., Reference Shi, Li, Deng, Li, Sun, Yuan, Song, Wang, Li and Li2014). Once cells are treated with various inducers, IκB is degraded, and NF-κB p65 translocates into the nucleus after phosphorylation (Xiong et al., Reference Xiong, Xu, Tan, Han, Tang, Xu, Zeng and Guo2015). Eventually, this promotes the transcription of target genes, such as TNF-α, IL-6, and IL-1β (Shi et al., Reference Shi, Li, Deng, Li, Sun, Yuan, Song, Wang, Li and Li2014). To further characterize the inhibitory mechanism of BBR in cytokine production, we examined the effect of BBR on the NF-κB pathway. In this study, we found that exposure of ruminal epithelial cells to LPS could increase phosphorylation of IκBα and NF-κB p65 and promote the translocation of NF-κB p65 to the nucleus. When cells were treated with different concentrations of BBR, the phosphorylation of IκBα and NF-κB p65 was inhibited in a dose-dependent manner. These results suggested that BBR suppressed the production of pro-inflammatory cytokines by preventing the activation of NF-κB pathway in ruminal epithelial cells. Furthermore, over-activation of MAPK-mediated inflammatory pathway also plays a key role during inflammatory responses (Di et al., Reference Di, Huang and Ho2011). We also examined the effect of BBR on the activation of p38, ERK and JNK in the MAPK pathway. Our results showed that BBR significantly inhibited the phosphorylation of JNK and p38MAPK but not that of ERK, which suggested that BBR could significantly inhibit the over-activation of JNK and p38MAPK induced by LPS. Taken together, our results clearly demonstrated that BBR suppressed pro-inflammatory cytokine production by preventing the activation of TLR4-mediated NF-κB and MAPK pathways induced by LPS in ruminal epithelial cells.
In conclusion, this study demonstrates that the anti-inflammatory effect of BBR in LPS-induced inflammation in ruminal epithelial cells may be due to its ability to inhibit the TLR4-mediated NF-κB and MAPK signaling pathways, which further inhibits the expression of TNF-α, IL-1β, and IL-6. Therefore, BBR may be a promising therapeutic reagent for rumen inflammation in cows with SARA.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029919000323
Acknowledgements
This work was supported by the National key research and development program (Beijing, China; grant no. 2016YFD0501206), the National Natural Science Foundation of China (Beijing, China; grant no. 31460681, 31472247, 31672621 and 31772810), the Jilin province education department project (Jilin, China; grant no. 440020032059).
Conflict of interest
The authors declare no conflicts of interest.
