Cocultivation of Chinese prescription and intestine microbiota:

IntroductionIrritable bowel syndrome (IBS) is a common and recurrent disease, with an internationally pooled prevalence of 12.41% and limited treatments (1, 2). Diarrhea-predominant irritable bowel syndrome (IBS-D), as the major type of IBS, is characterized by perennial abdominal pain and diarrhea (3), with complex and diverse pathogenesis (4–6). Although its etiology remains unclear, microbial factors play key roles in IBS pathophysiology (7). Both the structure and function of the intestinal microbiota of IBS-D patients are intensively disturbed (8, 9). The multifactorial pathophysiology of IBS-D suggests multiple therapeutic approaches, such as altering intestine microbiota, visceral hypersensitivity, intestinal permeability, gut-brain interaction and psychological strategies (10). Therefore, complementary and alternative medicines such as traditional Chinese medicine (TCM) featuring synergistic effects may show special activities for IBS-D (11).Few investigations have shown direct interactions between TCM formulas and gut microbiota. It is well known that oral administration is the major routine of TCM that inevitably interacts with the intestinal microbiota. The slight alterations of the construction and function in gut microbiota can significantly change the decomposition, transformation, and absorption of complex ingredients in Chinese herbs. Hence, directly detecting the potential molecular interactions between the intestinal microbiota and TCM herbs is pivotal for probing their interactive mechanisms. For instance, Si-Jun-Zi Decoction (SJZD) has been extensively used for IBS-D and other intestinal complaints for hundreds of years (11, 12). Unfortunately, the underlying mechanisms of SJZD via intestinal microbiota against IBS-D have not been fully described.The ex vivo fermentation system is a promising platform for exploring the direct interactions between TCM formulas and intestinal microbiota. Accumulating publications have demonstrated that gut microbiota can transform ingredients in TCM herbs into diverse metabolites that show different bioavailability, bioactivity and/or toxicity (13), which can intensively impact the health and disease of mammalian hosts (14). A considerable amount of research has been performed to explore the interactions between intestinal microbiota (always from healthy volunteers) and the ingredients of certain TCM herbs (15, 16). However, few studies have explored the precise interactions of intestinal microbiota derived from particular patients and relevant TCM formulas (17, 18). Originally from long-term successful clinical practice, TCM formulas consisting of several herbs create a much more complicated mechanism because different active ingredients can work together to produce a more desired health effect or can cancel out the negative effects associated with a single herb, thereby minimizing side effects and retaining only the desired effect.In the present work, an ex vivo cocultivation system will be established to determine the direct interactions of SJZD with the intestinal microbiota derived from IBS-D patients (19). To simulate the interactive regulation process between IBS-D intestine flora and effective TCM formulas, this anaerobic fermentation platform is of scientific significance for elucidating the interactive molecular mechanisms of a large number of TCM prescriptions and sparks a revolution in drug discovery based on Chinese formulas (20).Materials and methodsCollection and preparation of samplesThis work was approved by the Ethics Committee of the Chengdu University of TCM. Each participant signed an informed consent form before the experiment. Five representative IBS-D patients (group IBS-D) were diagnosed and recruited according to TCM standards (ZY/T001.9~001.9.94 & GB/T16751.2~1997) for the screen of Spleen-deficiency syndrome and Rome III diagnostic criteria for IBS-D identification. Five age-matched healthy subjects were simultaneously enrolled as normal controls (NC group). All participants had a routine diet before sample collection. None of the volunteers had taken antibiotics for the last three months. All fecal samples were collected under sterile rule. A solution for sample preservation and transportation, composed of KH2PO4 (0.45%), Na2HPO4 (0.6%), Tween-80 (0.05%), and agar (0.1%), was prepared and applied for temporary storage and transit of collected samples.Preparation of SJZD solutionAll herbs of SJZD formula were purchased from the Affiliated Hospital, Chengdu University of TCM. It consists of six herbs: Codonopsis Tangshan Oliv., Atractylodes macrocephala Koidz., Poria cocos (Schw. Wolf, Glycyrrhiza uralensis Fisch, Citrus reticulata Blanco, Zingiber oj-jicinale Rosc., 30 g for each herb. This formula was prepared under the manufacturing rule of TCM decoction (21). The applied solution of SJZD was adjusted to 1 g/ml (W/V).Coculturation of SJZD with intestine microbiotaReagentsTween-80 (Kemiou Chemical reagent, Tianjin, China), vitamin K1 (Solarbio Biotechnology, Beijing, China), and protohemin (Meilun Biotech, Dalian, China) were used in anaerobic culture. The culture medium, named General Anaerobic Medium (GAM) (Hopebiol Biotechnology, Qingdao, China), was composed of peptone 5.0 g, proteose peptone 5.0 g, enzymatically digested soybean meal 3.0 g, serum powder 10.0 g, beef extract 2.2 g, yeast extract 2.5 g, liver infusion 1.2 g, soluble starch 5.0 g, dextrose 0.5 g, sodium chloride 3.0 g, monopotassium phosphate 2.5 g, L-tryptophan 0.2 g, L-arginine 1.0 g, sodium thioglycollate 0.3 g, and L-cysteine monohydrochloride 0.3 g, and water was added up to 1000 mL. pH 7.3 was adjusted, sterilized at 121 °C for 20 min, and stored at 4°C for culture. The culture solution (KH2PO4 4.5 g, Na2HPO4 6 g, Tween-80 0.5 g, agar 1 g, added distilled water to 1000 ml) was mixed with GAM 3:7 (V/V) to obtain the transfer solution.Preparation of intestinal microbiotaApproximately 1 g of fecal sample was collected from each volunteer. The fecal sample was immediately put into a tube with 2 ml of the solution for sample preservation and transportation as described above. After slight mixing at 4°C for 5 min, 0.2 ml of the mixture was removed from each sample. The representative intestinal microbiota was then obtained by putting together five mixtures of groups IBS-D or NC and stored at -80°C.CocultivationThe representative intestinal microbiota derived from the IBS-D and NC groups were resuscitated in GAM (1:9, V/V) at 37°C. A total of 10 ml of gut microbiota was mixed with 90 ml of drug-containing GAM (1:4, V/V) and cultured in a 250 ml flask located in an anaerobic airbag at 37°C for 35 h. NC0, NC6, NC12, NC24 and NC35 represent the coculture mixtures from group NC and sampled at 0, 6, 12, 24, and 35 h postincubation, respectively, while IBSD0, IBSD6, IBSD12, IBSD24, and IBSD35 represent those of group IBS-D. Three duplicated samples of each group were performed and centrifuged at 4000 g and 4°C for 10 min. The supernatant was collected, and three samples at each time point were equally mixed together and stored in a -80°C refrigerator.16S rRNA gene sequencingCetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) were used to extract the total DNA from each sample of fermentation solution. The V3 and V4 regions of the 16S rRNA gene were amplified with a barcode (22) by a high-fidelity PCR master mix (New England Biolabs). The PCR products were then purified with a GeneJET gel extraction kit (Thermo Scientific). Sequencing libraries were generated using the NEB Next® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations, and index codes were added. Sequencing data were analyzed by a quantitative kit (Kapa Biosystems, KK4824) using an Illumina MiSeq platform for paired-end sequencing. The operational taxonomic units (OTUs) were selected through open reference OTU selection. Using LEfSe software, the LDA score was set to 4, and the community structure differences of the samples were analyzed. Metastatic analysis was carried out by R (Version 2.15.3) to analyze the differences between groups at each classification level, and the p value was obtained. Referring to Benjamin and
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