Effect of berberine on LPS-induced expression of NF-κB/MAPK signalling pathway and related inflammatory cytokines in porcine intestinal epithelial cells.

Berberine is an alkaloid extracted from medicinal plants such as Coptis chinensis and Phellodendron chinense. It possesses anti-inflammatory, anti-tumour and anti-oxidation properties, and regulates Glc and lipid metabolism. This study explored the mechanisms of the protective effects of berberine on barrier function and inflammatory damage in porcine intestinal epithelial cells (IPEC-J2) induced by LPS. We first evaluated the effects of berberine and LPS on cell viability. IPEC-J2 cells were treated with 5 μg/ml LPS for 1 h to establish an inflammatory model, and 75, 150 and 250 μg/ml berberine were used in further experiments. The expression of IL-1β, IL-6 and TNF-α was measured by RT-PCR. The key proteins of the NF-κB/MAPK signalling pathway (IκBα, p-IκBα, p65, p-p65, c-Jun N-terminal kinase (JNK), p-JNK, p38, p-p38, ERK1/2 and p-ERK1/2) were detected by Western blot. Upon exposure to LPS, IL-1β, IL-6 and TNF-α mRNA levels and p-IκBα p-p65 protein levels were significantly enhanced. Pre-treatment with berberine reduced the expression of inflammatory factors and was positively correlated with its concentration, and dose dependently inhibited the expression of IκBα, p-IκBα, p-p65, p-p38 and JNK. These results demonstrated that pre-treating intestinal epithelial cells with berberine was useful in preventing and treating diarrhoea induced by Escherichia coli in weaned pigs.

Introduction

Weaned piglets are susceptible to invasion by pathogenic micro-organisms due to immature intestinal immune function and the weakening and disappearance of maternal antibody.[1] Therefore, during the transition to active immunization, weaning piglet diarrhea caused by enterotoxigenic Escherichia coli is prone to occur.[2] LPS is a major glycolipid within the outer membrane of most Gram-negative bacteria and, depending on its structure, can be an endotoxin representing an important initiation factor mediating endotoxaemia. Various studies have shown that it can induce the synthesis and release of a variety of cytokines in the host through its binding receptors or regulatory proteins, trigger microcirculation disorders in the intestine and the whole body, and even diffuse intravascular coagulation. It can also activate epithelial cells to release a large number of cytokines and inflammatory mediators which exacerbate the inflammatory response and inflammatory damage to local tissues.[3-5] LPS is the best characterised PAMP which is recognised by the specific PRR TLR4.[6] TLR4 plays a crucial role in innate immunity and inflammation, triggering the activation of NF-κB and activator protein-1 signal transduction pathways. NF-κB plays a crucial role in regulating the transcription of genes related to innate immunity and inflammation responses, in turn increasing the expression of various genes such as IL-1β, IL-6 and TNF-α.[7] NF-κB activation is regulated by MAPK.[8]

Berberine is a yellowish phytochemical isoquinoline alkaloid ingredient isolated from rhizomes of Coptis chinensis and Phellodendron chinense. The application of berberine in Traditional Chinese Medicine has a long history and has been proven to have a variety of pharmacological and biological effects, including anti-cancer, anti-hyperlipidaemia, antibacterial, anti-inflammatory and antioxidant properties.[9,10] Berberine can effectively treat intestinal inflammation, inhibit the destruction of intestinal epithelial cell integrity induced by dextran sodium sulphate, inhibit the expression of chemokines in blood monocytes and macrophages induced by LPS, and intestinal inflammation factor secretion.[11,12] In order to explain the anti-inflammatory mechanism of berberine action in preventing and treating intestinal inflammation induced by LPS, this study examined the effect of berberine on the expression of inflammatory cytokines and key proteins in the NF-κB/MAPK signalling pathway after LPS-induced stimulation of porcine intestinal epithelial cells.

Methods

Chemicals and reagents

Berberine hydrochloride (≥ 98% purity, dissolved in PBS, passed through a 0.22 μm filter membrane) was obtained from Must (Chengdu, PR China). LPS (Escherichia coli O55:B5) and 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St Louis, MO). DMEM/F12 medium, FBS and EDTA-trypsin were obtained from Gibco Invitrogen (Carlsbad, CA). A PrimerScript RT reagent kit and SYBR Green PCR kit were purchased from Takara Bio (Dalian, PR China). The Abs against IκΒα, NF-κB p65, p-p65, p38, p-p38, c-Jun N-terminal kinase (JNK), p-JNK, ERK1/2, p-ERK1/2, anti-rabbit and anti-mouse Ab were purchased from Cell Signaling Technology (Beverly, MA). A bicinchoninic acid (BCA) protein assay kit and penicillin-streptomycin were purchased from Solarbio (Beijing, PR China). Unless otherwise specified, all other reagents were analytical grade.

Cell line and culture

IPEC-J2 cells were obtained from Southwestern University’s Biological Feed and Molecular Nutrition Laboratory. Cells were cultured in DMEM/F12 supplemented with 10% newborn calf serum, 1% penicillin-streptomycin at 37°C under an atmosphere of 5% CO2. In all experiments, cells were acclimated for 24 h and then pre-treated with different concentrations of Berberine dissolved in DMEM/F12 basic medium for 4 h prior to LPS (5 μg/ml, dissolved in DMEM/F12 basic medium) stimulation. After 1 h of stimulation, IPEC-J2 cells were collected for further determination.

Cell viability evaluation by MTT assay

MTT assay was used to evaluate the effect of berberine and LPS on cell viability. Briefly, the IPEC-J2 cells were cultured on a 96-well plate at a density of 5 × 104 cells per well. After 24 h, cells were treated with various concentrations of berberine (25, 50, 75, 125, 150, 200 and 250 μg/ml) for 24 h. Subsequently, 10 μl of 5 mg/ml MTT reagent was added to each well and incubated at 37°C under an atmosphere of 5% CO2 for 4 h. After removing the supernatant, 100 μl DMSO were added to dissolve the crystals. Then, the absorption values were measured at 490 nm on the enzyme-labelled instrument. Cell viability at each concentration was calculated separately as follows: % cell viability = (A drug group–A blank group)/(A control group–A blank group)×100%. The optical density in the control group cells (no berberine and LPS treatment) was considered 100% viability.

RNA isolation, cDNA synthesis and quantitative RT-PCR

The mRNA expression of IL-1β, IL-6 and TNF-ɑ were measured by quantitative RT-PCR, and β-actin was used as an internal control for mRNAs. IPEC-J2 cells were cultured on a six-well plate at a density of 5 × 106 cells per well for 48 h, and then divided into five groups as follows: (a) control group (con), where cells were kept in DMEM without FBS; (b) model group (mod), where cells were cultured in DMEM without FBS containing 5 μg/ml LPS; (c) low-dose berberine-treated group (low); (d) middle-dose berberine-treated group (mid); and (e) high-dose berberine-treated group (hig). Total RNA was isolated from the IPEC-J2 cells with Trizol reagent according to the manufacturer’s instructions and reverse transcribed into cDNA using the PrimerScript RT reagent kit. PCR amplification was performed with a SYBR Green PCR kit in a Bio-Rad RT-PCR machine in triplicate independently. The following primer sequences were used: IL-1β F: 5′-CGTGCAATGATGACTTTGTCTGT-3′, R: 5′-AGAGCCTTCAGCATGTGTGG-3′; IL-6 F: 5′-GCTGCAGTCACAGAACGAGT-3′, R: 5′-GGACAGGTTTCTGACCAGAGG-3′; TNF-α F: 5′-TTGAGCATCAACCCTCTGGC-3′, R: 5′-GGCATACCCACTCTGCCATT-3′. PCR parameters were: 3 min 95°C for one cycle, 5s 95°C, annealing temperature 30s for 40 cycles. Reaction specificity was controlled by post-amplification melting curve analyses. The expression fold-changes were analysed by the 2−ΔΔCt relative quantitative method.

Protein expression analysis by Western blotting

The protein levels of IκBα, p65, p-p65, p-IκBα, p38, JNK, p-JNK, p-p38, ERK1/2 and p-ERK1/2 were detected by Western blot analysis. In brief, the total proteins were extracted as follows. Cells were washed with ice-cold phosphate buffer and lysed in ice-cold lysis buffer for 30 min. The concentration of protein was determined using a BCA protein assay kit according to the manufacturer’s instructions. Equal amounts of protein were separated by 8% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were incubated with blocking buffer (5% w/v BSA in TBS containing 0.1% Tween-20) for 1 h at room temperature, and incubated with a primary Ab overnight at 4°C. After washing with TBST, the membranes were incubated for 1 h at room temperature with secondary Abs. The membranes were then detected by the electrochemiluminescence plus Western blotting detection system and quantified by Image Lab v5.0 (Bio-Rad, Hercules, CA).

Statistical analysis

Correlations among the markers of NF-κB and MAPKs were determined using Pearson’s correlation coefficient. Comparisons among the experimental groups were made using ANOVA, and differences between specific groups were determined using the Duncan’s multiple range test. Results are expressed as the mean ± SD in each group. A P value of < 0.05 was considered as significant, and a P value of < 0.01 was considered as extremely significant. All statistical analyses were conducted using IBM SPSS Statistics for Windows v26.0 (IBM Corp., Armonk, NY).

Results

Effects of different concentrations of berberine on the proliferation of porcine intestinal epithelial cells

First, we examined the potential cytotoxicity of berberine by the MTT assay after incubating cells for 24 h in the absence of LPS (Figure 1). The cell viability of IPEC-J2 cells in the 25 and 50 μg/ml berberine solution groups was significantly higher than that of the control group (P < 0.05). Cell viability of IPEC-J2 cells in 75, 100, 125, 150, 200 and 250 μg/ml berberine solution groups was extremely significantly higher than in the control group (P < 0.01). It peaked in the 150 μg/ml group and then decreased. Therefore, 75, 150 and 250 μg/ml were selected as the low, middle and high concentrations of berberine for subsequent experiments.

Figure 1.

Effect of berberine on the viability of IPEC-J2. The cells were pre-treated with berberine (25, 50, 75, 125, 150, 200 and 250 μg/ml) for 24 h. Cell viability was determined via 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay, and relative cell viability was calculated by compared to the control group.

Berberine suppressed pro-inflammatory cytokine expression in LPS-simulated IPEC-J2 cells

As shown in Figure 2, LPS treatment for 1 h highly significantly enhanced IL-1β, IL-6 and TNF-α mRNA levels in IPEC-J2 cells (P < 0.01). The expression of IL-6 and TNF-α mRNA in the low-dose berberine group was significantly higher than that in the control group (P < 0.05). The expression of TNF-α in the middle-dose group was significantly higher than that in the control group, and there was no significant difference between the other groups and the control group (P > 0.05). Compared to the model group, there was a significant decrease in each drug group. Therefore, these findings showed that berberine significantly inhibited the LPS-induced expression of pro-inflammatory cytokines.

Figure 2.

Berberine suppressed pro-inflammatory genes expression in LPS-stimulated IPEC-J2. Changes in gene expressions of (a) IL-1β, (b) IL-6 and (c) TNF-α. Cells were pre-treated with berberine for 4 h before incubation with 5 μg/ml LPS for 1 h. *P < 0.05); **P < 0.01, both compared to the control. #P < 0.05; ##P < 0.01, both compared to the model.

Effect of berberine on the NF-κB/MAPK signalling pathway in IPEC-J2 cells

Effect of berberine on protein expression in the NF-κB signalling pathway in IPEC-J2 cells

As shown in Figure 3, after exposure to LPS for 1 h, the protein expression levels of IκBα, p-p65 and p-IκBα in IPEC-J2 cells were significantly higher than those in the control group. However, berberine (75, 150 and 250 μg/ml) pre-treatment for 4 h dose dependently inhibited LPS-induced IκΒα, p-p65 and p-IκΒα expression. The protein expression levels of IκΒα, p-IκBα and p-p65 in cells of the high-dose berberine group were significantly or extremely significantly lower than those in the model group (P < 0.05 and P < 0.01, respectively). The low- and middle-dose berberine groups were extremely significantly lower than the model group.

Figure 3.

Berberine inhibited protein expression of the NF-κB signalling pathway in LPS-stimulated IPEC-J2 cells. Changes in protein expressions of (a) IκΒα/p- IκΒα and (b) p65/p-p65. Cells were pre-treated with berberine for 4 h before incubation with 5 μg/ml LPS for 1 h. *P < 0.05); **P < 0.01, both compared to the control. #P < 0.05; ##P < 0.01, both compared to the model.

Effect of berberine on the MAPK signalling pathway in IPEC-J2 cells

As shown in Figure 4, after 1 h of LPS treatment of IPEC-J2 cells, the protein expression levels of p38, JNK, p-JNK, p-p38, ERK1/2 and p-ERK1/2 were significantly or extremely significant increased (P < 0.05 or P < 0.01). Pretreated with low, middle and high doses of berberine, the protein expression levels of p38, p-p38 and p-JNK in IPEC-J2 decreased compared to the model group. The protein expression levels of p-JNK, p-p38 and p-ERK1/2 in the middle- and high-dose groups were extremely significantly lower than those in the model group (P < 0.01) and significantly or extremely significantly lower than those in the control group (P < 0.05 and P < 0.01, respectively).

Figure 4.

Berberine inhibits protein expression of the MAPK signalling pathway in LPS-stimulated IPEC-J2 cells. Changes in protein expressions of (a) p38/p-p38, (b) JNK/p-JNK and (c) ERK 1/2/p-ERK 1/2. Cells were pre-treated with berberine for 4 h before incubation with 5 μg/ml LPS for 1 h. *P < 0.05; **P < 0.01, both compared to the control. #P < 0.05; ##P < 0.01, both compared to the model.

Pearson correlation coefficient between phosphorylation/total proteins of key proteins in the NF-κB/MAPK signalling pathway

The results summarised in Table 1 indicate that there was a positive correlation between phosphorylation/total proteins of key proteins in the NF-κB/MAPK signalling pathway. Except for IκBα/JNK, IκBα/p65 and p38/JNK that all showed an extremely high correlation, IκBα/P38 and IκBα/ERK1/2 showed a strong correlation, as did P38/ERK1/2. Similarly, p38/ERK1/2 and p38/JNK were strongly correlated.

Table 1.

Pearson’s correlation coefficients between phosphorylation of key proteins of the NF-κB/MAPK signalling pathway and total protein ratio.

	IκBα	p65	p38	ERK 1/2	JNK
IκBα	1	0.829**	0.797**	0.729**	0.811**
p65		1	0.682*	0.599*	0.912**
p38			1	0.737**	0.799**
ERK 1/2				1	0.625*
JNK					1

*P < 0.05; **P < 0.01. 0.8–1.0 extreme correlation, 0.6–0.8 strong correlation, 0.4–0.6 moderate correlation, 0.2–0.4 weak correlation, 0.2–0.0 very weak correlation or no correlation.

JNK: c-Jun N-terminal kinase.

Discussion

E. coli is the main cause of yellow scour and oedema in newborn piglets. LPS is one of the main components of the cell envelope of E. coli, which can trigger a strong inflammatory response in porcine intestinal epithelial cells and release many pro-inflammatory cytokines,[13] causing severe intestinal inflammation in pigs. Berberine is a quaternary ammonium base extracted from natural plants such as Coptis chinensis and Phellodendron chinense, and it has a wide range of pharmacological effects, such as antibacterial, antiviral, anti-inflammatory, analgesic and anti-cancer. It is commonly used for the clinical treatment of gastroenteritis, bacterial diarrhoea, intestinal infections and conjunctivitis.[14]

The MTT assay is a commonly used method to estimate the metabolic activity of living cells to detect proliferation, differentiation and cell metabolism. This test is based on the reduction of MTT by succinate dehydrogenase in living cells to form blue crystals, which can be quantified spectrophotometrically. Under appropriate optimised conditions, the absorbance obtained is directly proportional to the number of cells.[15] Numerous studies have shown that LPS promotes cell proliferation by activating NF-κB,[16-18] with 1 and 10 μg/ml LPS effectively inducing the proliferation and secretion of inflammatory cytokines in IPEC-J2.[19] Therefore, we selected 5 μg/ml LPS as a concentration for inducing an IPEC-J2 inflammatory response. At the same time, different concentrations of Berberine (25, 50, 75, 125, 150, 200 and 250 μg/ml) could improve the cell viability of IPEC-J2 cells, that is, effectively promote their proliferation and growth, and help to repair the intestinal mucosa. We selected 75, 150 and 250 μg/ml as the low, middle and high concentrations, respectively, of berberine for subsequent studies.

IL-1β, IL-6 and TNF-α are pro-inflammatory cytokines which can induce a severe inflammatory immune response in tissue cells. IL-1β can promote cell growth and differentiation and apoptosis, and induce inflammatory responses. IL-6 can induce lymphocyte differentiation and proliferation, and eliminate pathogenic micro-organisms.[20] IL-6 also protects against endotoxin-mediated injury and death by down-regulating the expression of inflammatory mediators.[21] TNF-α can promote the expression of vascular endothelial cells at the infection site, stimulate IL and chemokines, induce many leucocytes to aggregate in the inflammation site and promote the removal of bacterial in the inflammation site,[22] and represents a drug target for treating inflammatory diseases.[23] The above pro-inflammatory cytokines have important immune-regulatory effects in the inflammatory response of tissue cells.

This study shows that LPS could induce increased mRNA expression of IL-1β, IL-6 and TNF-α in IPEC-J2 cells, and shows that LPS is one of the major toxins of E. coli released into the intestine, which could cause increased expression of inflammatory cytokines in IPEC-J2 cells and inflammatory reactions, consistent with results reported in the literature.[24,25] However, after pre-treatment with berberine, the expression of inflammatory cytokines in IPEC-J2 cells did not increase after continuous action of LPS, with the best effect in the high-dose group. These results indicated that berberine can inhibit the expression of inflammatory cytokines in IPEC-J2 cells induced by LPS, and can regulate the intestinal inflammatory response. It is helpful to explain the mechanism of berberine in preventing and treating PWC.

NF-κB is a family of transcription factors widely present in various cells, which can regulate the cell immune response, inflammatory response and apoptosis, and other related gene transcription. It can be activated by a variety of stimulating factors (LPS, TNF-α, IL-1β) and induce expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α). NF-κB is normally sequestered in the cytoplasm of non-stimulated cells by inhibitors of NF-κB, in which IκBα is the principal subunit.[26,27] The activation of NF-κB usually involves the phosphorylation of IκBα followed by its degradation.[28] Consistently, we observed that after adding LPS, IκBα, p-IκBα and p-65 increased sharply. On the contrary, after pre-treatment with berberine, the expression of IκBα, p-IκBα and p-65 was dose dependently and significantly reduced.

MAPK is a signal transduction module in eukaryotic cells which can mediate extracellular signals (hormones, cytokines and growth factors) into intracellular responses, and regulate the processes of cell growth, differentiation, migration and inflammation.[29] The MAPK pathway consists of a three-layer kinase module in which MAPK is activated after being phosphorylated by MAPK kinase (MAPKK), and is activated again when phosphorylated by MAPKK.[30] There are four major MAPK groups: ERK, ERK5, JNK and p38.[31] Inflammatory cytokines, growth factors and cellular stress can activate the MAPK pathway and regulate the expression of downstream inflammatory cytokines. This study showed that berberine could effectively inhibit the protein expression levels of p-ERK1/2, p-JNK, p38 and p-p38 in the MAPK signal pathway in IPEC-J2 induced by LPS. Berberine showed the best effect in the middle- and high-dose groups.

Previous research has found that p38, ERK, JNK and NF-κB play central roles in the regulation of various inflammatory responses.[32] The interaction between these pathways usually determines the final biological response after inflammatory mediator stimulation.[33] TNF-α and IL-1β stimuli induce the simultaneous activation of NF-κB, JNK and p38 kinase, and a connection between these MAPK pathways and NF-κB activation has now been established at the molecular level.[34] Studies have shown that specific inhibitors of p38 MAPKs can block LPS-induced adhesion and NF-κB activation.[35] However, p38 MAPK inhibitors do not affect phosphorylation of NF-κB subunits, nuclear translocation and DNA binding of NF-κB.[36] This indicates that NF-κB and p38 MAPK are activated by separate effector pathways and converges downstream of the nucleus.[34] Our study could prove a correlation between NF-κB and MAPK signalling pathways. Phosphorylation of key proteins in the NF-κB/MAPK signalling pathways induced by LPS resulted in a downstream pro-inflammatory factor expression. Berberine pre-treatment could effectively inhibit LPS-induced activation of the NF-κB/MAPK signalling pathway in IPEC-J2 cells, and inhibit the production of downstream inflammatory factors. Therefore, berberine may prevent the intestinal inflammation induced by LPS through inhibiting the interaction between the NF-κB/MAPK signalling pathway and related inflammatory factors.

Conclusion

In summary, our results demonstrate that berberine has potential inhibitory properties on an LPS-induced inflammatory response. Its mechanism is related to inhibiting the mRNA expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in IPEC-J2 cells, and the phosphorylation and expression of key proteins of the NF-κB/MAPK signalling pathway activated by LPS. These results suggested that berberine may be an effective natural product for preventing swine intestinal inflammation disease associated with LPS.