Dehydroandrographolide enhances innate immunity of intestinal tract through up-regulation the expression of hBD-2
- Wen-Bi Xiong†1,
- Zhen-Jun Shao†1,
- Yao Xiong1,
- Jian Chen1,
- Yun Sun1,
- Ling Zhu1Email author and
- Li-Ming Zhou1Email author
© Xiong et al. 2015
Received: 19 April 2015
Accepted: 15 July 2015
Published: 30 July 2015
Dehydroandrographolide (DA) is one of major active components in the well-known oriental herbal medicine Andrographis paniculata (Burm.f) Nees which belongs to the Acanthaceae family. DA is used for the treatment of infections in China. However, DA has not been found to significantly inhibit bacterial and viral growth directly. The current study investigates the effect of DA on the expression of human β –defensin-2 (hBD-2) in human intestinal epithelial cells and the possible signaling pathways.
Human intestinal epithelial HCT-116 cells were incubated with 1–100 μM DA for 2–24 h. RT–PCR and Western blot were used to assess the expression of hBD-2. The specific inhibitors were used and the levels of phosphorylation of signaling molecules were detected for dissecting the signaling pathways leading to the induction of hBD-2.
MTT assay showed there was no obvious cytotoxicity for HCT-116 cells by 1–100 μM DA treatment. RT-PCR and Western blot assays showed that DA (1–100 μM) could up-regulate the expression of hBD-2, and the effect lasted longer than 24 h. By using SB203580 and SB202190 (inhibitors of p38), the enhancement of hBD-2 expression were significantly attenuated. However, inhibitor of ERK and inhibitor of JNK could not block the effect of DA. Furthermore, Western blot found activation of p38 but not ERK and JNK in DA-treated HCT-116 cells.
The results suggested that DA enhanced innate immunity of intestinal tract by up-regulating the expression of hBD-2 through the p38 MAPK pathways.
KeywordsInnate immunity Dehydroandrographolide hBD-2 Antimicrobial peptide defensin p38 MAPK
Andrographis paniculata (Burm. f) Nees, which belongs to the Acanthaceae family, is an oriental herbal medicine that has been widely used in China, India, and other Southeastern Asian countries for hundreds of years due to its significant antipyretic and anti-inflammatory effect. In China, A. paniculata is often used for treatment of chronic or repeated onset infection and inflammation, especially for the upper respiratory tract infection and gut diarrhea [1–3]. Both andrographolide (Androg) and dehydroandrographolide (DA) are main active components in A. paniculata, which have been demonstrated to contribute anti-infectious property of the A. paniculata. Contemporary laboratory tests have exhibited their anti-inflammatory, antipyretic, antimicrobial, antiviral and immunostimulant capability [4–6]. In China, Androg and DA are called “Natural Antibiotics” . However, the mechanism has not been fully clarified and Androg and DA have not been found to significantly inhibit bacterium and virus growth directly . Our recent work demonstrated that DA could enhance innate immune function by up-regulating the expression of human β –defensin (hBD-1) of human lung epithelial cells .
Innate immunity plays a very important role for mucous membranes in defense against invading pathogenic microbes. The human gut harbors trillions of microbes which are essential for mediating physiology, metabolism and host immune responses. In normal condition, the microbes could coexist peacefully with the body owing to the innate immunity despite the intestinal mucosal surface acting as a primary barrier . Hence, it is a good choice to enhance intestinal innate immune function for treatment of intestinal infection rather than using antibiotic for less antibiotic resistance and dysbacteriosis, and it is an alternative strategy to look for active ingredients to enhance the body's innate immunity from natural products such as ellagic acid , paeoniflorin , avocado sugar  etc.
Defensins, one of the 3 major antimicrobial peptides (AMPs) families, are important moleculars in innate immune system. Defensins are small (Mr 2 000 ~ 6 000 Da), cationic peptides containing disulfide bonds, so can interact with the membrane of invading microbes and active against many Gram-negative and Gram-positive bacteria, fungi, and enveloped viruses . According to the position of their disulfide bonds human defensins are further classified as α- and β-defensins. Human β-Defensin (hBD) has six members which are expressed by many types of epithelial cells, including enterocytes. hBD-1, firstly discovered in 1995, is an important antimicrobial peptide against infection in human prostate, kidney and urogenital tract luminal epithelium . hBD-2, discovered in 1997 , is the first discovered inducible human antimicrobial protein. Moreover, it was shown that hBD-2 had a broad spectrum of antimicrobial activity in vitro . Therefore, in this study, we investigated the role of DA in hBD-2 expression in HCT-116 intestinal epithelial cells and the possible mechanism.
Materials and methods
Cell line and culture
HCT-116 intestinal cells, a human cell line derived from the colonic epithelial cell line, were maintained at 37 °C in a humidified atmosphere of 95 % air and 5 % CO2 in RPMI 1640 medium (Gibco-BRL, Grand Island, NY). The medium mixture contains 10 % fetal bovine serum, 100 U/ml penicillin G and 100 μg/ml streptomycin. After reaching confluence, cells were seeded onto six well plates and incubated with DA. For all experiments, DA were added to cells on ice and incubated for 10 min, allowing DA to settle onto the cells, and then incubated at 37 °C in 5 % CO2 for the specified intervals. Cells treated with lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, Mo., USA) were employed as positive control. Vehicle control with equal concentration of DMSO was always included. HCT-116 intestinal cells were exposed to the specified concentrations of each inhibitor for 30 min prior to stimulation with DA.
3-(4,5-Dimethylthiazol-zyl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the effect of DA on cell viability and to determine the non-cytotoxic concentrations. Briefly, HCT-116 cells (105) were seeded on 96 well plates and incubated with DA at various concentrations (0, 1, 20, 40, 80 and 100 μM) for 48 h. Thereafter, the medium was changed and MTT (1 mg/ml) was added to each well and incubated in the CO2 incubator for 4 h. The viable cell number was directly proportional to the production of formazan crystals which, following adding isopropanol 100 μl to dissolve and the absorbance of each well was obtained using a Bio-Rad Microplate reader at wavelength of 490 nm.
Semi-quantitative RT-PCR was performed as described previously . Total RNA was isolated from cells using Trizol (BBI, Kitchener, Ont., Canada), and samples of total RNA were quantified by reading optical density at 260 nm. mRNA was reverse transcribed using a first strand cDNA synthesis kit (Fermentas, Glenn Burnie, Md., USA) according to the manufacturer’s instructions. One microgram of cDNA was amplified in the standard reaction mixture using 2× PCR MasterMix (TIANGEN, China) and the forward and reverse primers for hBD-2 and β-actin, an internal standard. The forward and reverse primers and product sizes were as follows: hBD-2 (255 bp) forward: 5′-CCAGCCATCAGCCATGAGGGT- 3′; reverse: 5′-GGAGCCCTTTCTGAATCCGCA- 3′. For β-actin (300 bp), forward: 5′-TCACCCACACTGTGCCCATCTACGA- 3′; reverse: 5′-CAGCGGAACCGCTCATTGCCAATGG- 3′. Amplification proceeded in a PCR Thermal Cycler (Takara PCR Thermal Cycler MP; Takara Bio, Otsu-shi, Japan) using 32 cycles consisting of 94 °C for 60 s, 62 °C for 30 s, and 72 °C for 120 s. The PCR products were verified by electrophoresis in a 1.2 % agarose gel and detected by ethidium bromide staining. Detectable fluorescent bands were visualized with an ultraviolet transill. The values of densitometry of the mRNA bands were acquired using ImageJ software (National Institutes of Health, USA) and then normalized with β-actin.
Western blot analysis
After cells were treated with DA or inhibitors, total cell extracts were prepared in ice-cold lysis buffer (150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mg/mL aprotinin, 1 mg/mL pepstatin, 1 mmol/L pervanadate,1 mmol/L EDTA, 1 % igepal, 0.25 % deoxycholic acid,1 mmol/L NaF, and 50 mmol/L Tris–HCl (pH 7.4)). The lysates were centrifuged at 12 000 r/min for 10 min, and the resulting supernatants were assessed with a Bio-Rad assay kit using bovine serum albumin as the standard. Equal amounts of protein (40 μg/lane) were separated by SDS PAGE and blotted onto 0.22 μm polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, Calif., USA). The membranes were blocked in TBST (Tris-buffered solution, pH 7.6, 0.05 % Tween 20) containing 5 % nonfat dried milk. Membranes were probed overnight at 4 °C with specific primary antibodies including anti-hBD-2, anti-p-p38 (Thr180/182), p38 and anti-β-actin (Santa Cruz, Calif., USA); anti-ERK1/2, anti-p-ERK1/2, anti-JNK, anti-p-JNK (Cell Signaling Technology Inc, USA) . After 3 washes, the blots were subsequently incubated with appropriate secondary antibodies with horseradish peroxidase (HRP, ZSGB-BIO) at room temperature for 1 h and developed in the ECL Western blot detection reagents (Beyotime, Jiangsu, China). The values of densitometry of the protein bands were acquired using ImageJ software (National Institutes of Health, USA) and then normalized with β-actin.
Results were presented as mean ± SD. Data were statistically evaluated using one-way analysis of variance (ANOVA) followed by Dunnett’s test by SPSS 13.0 software (SPSS, Chicago, USA). A P value < 0.05 was considered as statistically significant.
Cytotoxicity of DA on HCT-116 intestinal epithelial cells
Expression of hBD-2 mRNA
Expression of hBD-2 protein
Mechanism of hBD-2 expression induced by DA
Both DA and Androg are main active components in A. paniculata which are popular used for all kinds of infection and inflammation in Asia. They have the similar structure and both are labdane diterpenoids. The only difference is the dehydroxylation in C-14 for DA comparing to Androg (Fig. 1). Consequently, they have the similar actions in many conditions [18, 19]. We have found that both Androg and DA could enhance innate immunity by stimulate hBD-1 and hBD-2 expression in respiratory tract epithelial cells . However, it has remained unclear whether they have the same effect in intestinal epithelial cells. Therefore, we first added different concentrations of Androg and DA into culture HCT-116 cells, found DA had less cytotoxic effect for HCT-116 cells than Androg (IC50 DA >200 μM vs IC50 Androg 10.67 μM for HCT-116 cells 48 h). As the lower toxicity of DA, DA succinate (trade name: Yan-Hu-Ning Injection)) is more popular used for injection in clinical for the treatment of viral pneumonia and upper respiratory tract infections in China [21, 22]. Therefore we focused on observing the effect of DA on hBD-2 expression in HCT-116 cells in this study. We found the level of mRNA and protein of hBD-2 increased 11 and 20 times comparing with control respectively in DA treatment group at non-toxic concentration 100 μM. The effect of DA induction hBD-2 expression was maintained for 24 h, which longer than human lung epithelial cells expression hBD-1.
hBD-2 plays important roles in innate defence system at gastrointestinal (GI) tract mucosal surfaces. Unlike hBD-1 constitutively expression within GI tract and mostly remain stable during inflammation and infection, hBD-2 is induced upon pathogen [23–25]. hBD-2 is seldom expressed in normal colon but its expression is significantly increased in inflamed colonic epithelium . It has been shown that certain probiotic strains can induce the secretion of antimicrobial peptides (such as hBD-2) by intestinal cells and probiotics have already been used as “ecological treatments” in gastrointestinal infections such as diarrhoea, dysentery . At the same time, scientists pay attention to looking for defensins inducer from Chinese herbal medicines such as Astragalus membranaceus (Fisch.) Bunge., Coptis chinensis Franch, Houttuynia cordata Thunb., Isatis tinctoria and Lentinan, which will also appear as attractive strategies for these diseases. From our results, DA was proved to be a defensins inducer and would be beneficial for the rehabilitation of intestinal infection.
For the promoter region of the hBD-2 gene contains binding sites for AP-1, and AP-1 is a transcription factor that can be activated by different MAPK pathways, including the p38, ERK and JNK pathway , we investigated whether MAPK signalling pathways were involved in DA induced hBD-2 expression in human intestinal epithelial cells. The results showed that it was the specific inhibitors of p38 MAPK but not the inhibitors of ERK and JNK significantly attenuated DA-induced hBD-2 gene expression. When measured the p-p38 (p38 active form) we found the p-p38 increased in DA treated HCT-116 cells, however the p-ERK (ERK active form) and p-JNK (JNK active form) had no significant change. Our findings on the signal passway of DA induced hBD-2 expression in human intestinal epithelial cells are partial consistent with that of a previous study by Gan et al. , which reported that paeoniflorin, a substance came from herbal plant peony upregulates β-defensin-2 expression in human bronchial epithelial cell through the p38 MAPK, ERK, and NF-κB signaling pathways. Several studies revealed different inducer may induce hBD-2 expression through different signalling pathways in different cells. Bacteroides fragilis enterotoxin induces hBD-2 expression in intestinal epithelial cells via p38/NF-κB-dependent pathway . Escherichia coli Nissle 1917 induces hBD-2 expression in intestinal epithelial cells via NF-κB and AP-1 signalling pathways . Avocado sugar modulates the hBD-2 expression through toll-like receptor-2 and ERK/MAPK activation in human keratinocytes . As DA is a potential NF-κB inhibitor [31, 32], whether NF-κB is involved in hBD-2 expression in intestinal epithelial cells need to further confirm. It is still a question whether DA will lead to more serious inflammatory for upregulation hBD-2. Especially for those associated with a high level of hBD-2 gut inflammation such as Crohn’s disease [33, 34]. Immune mediators often have dual-direction regulation like the water extract of Houttuynia cordata Thunb  and paeoniflorin,which could upregulate β-defensin-2 expression in human bronchial epithelial cell at the same time downregulated β-defensin-2 expression in mice colonic mucosa with oxazolone-induced colitis . The existing evidences have described DA is a immune mediator. DA could significantly reduce the level of IL-1β, IL-6 and TNF-α in the bronchoalveolar lavage fluid of the LPS-induced acute lung injury mouse . DA could dose-dependently inhibited ovalbumin-induced increases in total and eosinophil counts, IL-4, IL-5, and IL-13 levels in lavage fluid in a mouse asthma model . Therefore, DA may not enhance inflammatory at the same time of hBD-2 inducing in GI tract.
In summary, we have demonstrated that exposure of intestinal epithelial cells to DA caused the activation of a signaling cascade involving p38 MAPK up-regulation expression of hBD-2 in intestinal epithelial cells. This is one main reason why DA could be used for intestinal infection.
human β –defensin-2
Reverse transcription polymerase chain reaction
Mitogen-activated protein kinase
Extracellular regulated protein kinases
c-Jun N-terminal kinase
Nuclear factor kappa-light-chain-enhancer of activated B cells
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