Introduction
The inulin, consisting of fructose units linked by β-(2→1) bonds and a terminal glucose by α-(1→2) bond, remains relatively intact after transition through the digestive system (Shoaib et al., 2016). It was demonstrated with effects of improving immune and promoting the proliferation of beneficial bacteria, which are related with their degree of polymerization and plants species (Moreno-Vilet et al., 2014; García Gamboa et al., 2018). All these benefits are related to body health through positively balancing gut microbiota via increasing the abundance of beneficial bacteria and reducing potential pathogens. The pathogens colonization were inhibited by the antagonism from organic acids or inhibitory peptides (García Gamboa et al., 2018). Inulin is also identified with anti-obesity, anti-diabetes, anti-inflammatory, anti-hypertension, anti-oxidative and anti-cancer activities, as well as exerts abilities to control inflammatory bowel disease, promote colonic absorption of minerals and stimulate the immune system (Karimi et al., 2015; Vogt et al., 2015; Ahmed and Rashid, 2017; Shang HM et al., 2018; Mazraeh et al., 2019). These activities support inulin a widely use as function foods on reducing cardiovascular disease, lowering blood urea and uric-acid levels (Karimi et al., 2015).
The roots of Condonopsis pilosula (Radix Codonopsis) has been used in Traditional Chinese Medicine (TCM) for hundreds of years. Three species of its plant sources are recorded in the Pharmacopoeia of the People’s Republic of China, including Codonopsis pilosula (Franch.) Nannf., C. pilosula Nannf. var. modesta (Nannf.) L. T. Shen and C. tangshen Oliv. (Chinese Pharmacopoeia Committee, 2020), for the treatment of nourishing the spleen and stomach and tonifying Qi of stomach. Modern pharmacological studies have uncovered Radix Codonopsis with effects like immunomodulation, anti-ulcer, lowering blood pressure, as well as intestinal flora modulation. It has also been widely used as nutritional supplement in China on homology as both medicine and food (Gao et al., 2018). The polysaccharide fraction is considered as a potential bioactive natural product possessing modulatory effect on immunity and intestinal microbiota (Fu et al., 2018a; Peng et al., 2018; Deng et al., 2019; Zou et al., 2019). Similar functions of polysaccharides from C. pilosula Nannf. var. modesta (Nannf.) L. T. Shen (CPP), have been reported in our previous studies, like decreasing the levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-10, elevating serum ileum IgG, intestinal secretory immunoglobulin A (sIgA), and the amount of Lactobacillus and acetic acid content in cecum on cyclophosphamide (CY)-induced immunosuppressed mice (Fu et al., 2018a). And inulin, a reserve carbohydrate of Radix Codonopsis, has also been identified with high proportion (62%, w/w) as the neutral composition of CPP, with degree of polymerization (DP) of 2–17, and exerted underlying prebiotic activity in vitro (Fu et al., 2018b). Therefore, we deduced that the inulin as the main bioactive component of CPP, acts on intestinal mucosal improvement and microbiota regulation. The active target organ is possibly the intestine due to its non-digested property and the conclusions in our experimental work mentioned above. Moreover, the fermentable properties of inulin are related to their chemical structure, especially Mw (Gibson and Delzenne, 2008). There is a possibility that certain short inulin could have been digested completely when it arrives specific intestinal positions, which is seldom proofed by study of inulin from natural herbs. It is consequently of interest to figure out its efficacy on anti-inflammation in different intestinal segments, which could be helpful for the clinical application of inulin on various intestinal inflammatory disease.
Therefore, in this study, the intestinal modulatory effects of inulin from C. pilosula was evaluated from aspects of intestinal mucosal immunity, anti-inflammatory and gut microbiota modulatory functions on immunosuppressed mice. Among them, a comparison of anti-inflammatory with different intestinal segments was investigated in order to find the target intestine location.
Materials and Methods
Materials and Chemicals
The roots of C. pilosula Nannf. var. modesta L. T. Shen were collected in October 2017 from Jiuzhaigou County (Tibetan Qiang Autonomous Prefecture of Ngawa, China), and identified by Yuan-Feng Zou, College of Veterinary Medicine, Sichuan Agricultural University. The roots were dried and pulverized to a fine powder, and the fructan was obtained by DEAE-sepharose gel chromatograph, and identified as inulin-type fructan (the fructan form C. pilosula, CPPF) (Fu et al., 2018b).
The cyclophosphamide (CY, C8650) was obtained from Solarbio technology Co., Ltd., (Beijing, China). The ELISA kits, including mouse IL-1β, tumor necrosis factor-α (TNF-α), sIgA and mucin 2 (Muc2), were purchased from Enzyme-linked Biotechnology Co., Ltd., (Shanghai, China). The acetic acid (71251), propionic acid (94425) and butyric acid (19215) standards were purchased from Sigma-Aldrich (St. Louis, MO, United States). The primeScript RT reagent kit (with gDNA Eraser, RR047A), the TB Green Premix Ex Taq II (Tli RNaseH Plus, RR821A) and Trizol RNA isolation reagent were obtained from TAKARA, Japan. All other chemicals, such as chloroform, isopropanol, etc., were of analytical grade, obtained from the Chengdu Kelong chemical factory (Chengdu, China).
Animal Care and Experimental Design
Sixty male specific-pathogen-free C57BL/6 mice (6–8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing China). They were maintained in a specific pathogen-free environment, where the temperature was 25 ± 2°C, with humidity of 60%. All mice were acclimatized for 7 days with an automatically-controlled 12 h light/dark cycle and free access to sterile food and distilled water.
Although CY is an alkylating drug used on cancer and autoimmune disease, it has been used as an immunosuppression-inducing agent due to its major side effect (Wang Z et al., 2019). In this study, a stable and classical CY-induced immunosuppression model was used to evaluate the recovery effect of CPPF on both intestinal immunity and microbiota modulation, according to several previous studies (Tang et al., 2018; Zhu et al., 2018; Wang Z et al., 2019). The mice were divided into five groups, 12 mice each. The immunosuppressed mice (4 groups) were given 60 mg/kg (0.1 ml/10 g body weight) CY for 3 days via intraperitoneal injection, once a day (Fu et al., 2018a). The normal mice were injected with 0.1 ml/10 g body weight saline as Control group. After 3 days of CY/saline treatment, the mice were administrated orally with freshly prepared CPPF (dissolved in saline) at dosage of 200, 100, 50 mg/kg (dosages were set in line with that of CPP in our previous study (Fu et al., 2018a) or saline (0.1 ml/10 g body weight) for 7 days in succession, as CY + CPPF-H, CY + CPPF-M, CY + CPPF-L and Control/CY groups, respectively. After 24 h of the last administration, all mice were euthanized with carbon dioxide followed by cervical dislocation, and the different segments of intestinal tissues, and the content of cecum were separated immediately and stored at −80°C.
Determination of the Intestinal Cytokines, sIgA and Muc 2
IL-1β and TNF-α secretions in intestine tissues (including jejunum, ileum, and colon), and sIgA, Muc2 in ileum, were determined using ELISA kits. The intestinal tissues were ground in liquid nitrogen and homogenized in saline (50 mg/ml). The supernatants after centrifugation at 2,862 g for 20 min, 4°C, were collected and determined according to the manufacturer’s instructions.
Quantitative Real-Time PCR
RNA extraction from jejunum, ileum and colon tissues (without content) and the quantitative real-time PCR (qRT-PCR) were performed as previously reported (Huang et al., 2014). Briefly, total RNA was extracted using Trizol reagent, and the quality and nucleic acid concentration were measured using spectrophotometer (NanoDrop 2000; Thermo Scientific, Shanghai, China). Reverse transcription was processed according to the manufacturer’s instructions (two-step). Quantitative real-time PCR was performed using the Bio-Rad CFX-96 system and TB Green Premix Ex Taq II kit. Gene expressions were normalized related to β-Actin, with primer sequences for SYBR Green probes of NGB obtained from Primer Bank: β-actin (No. 6671509a1), IL-1β (No.118130747c1) and TNF-α (No.133892368c2).
Bacteriological Analysis and the Determination of Short-Chain Fatty Acids
The intestinal digesta of cecum were isolated after last gavage, and divided into two parts stored at −20°C (for SCFAs determination) and −80°C (for the bacteriological analysis). The samples stored at −80°C were transported to Beijing Novogene Science and Technology Co., Ltd. under dry-ice cooling environment. The DNA was extracted using CTAB/SDS method, and diluted to 1 ng/μL using sterile water. The V4 region of 16S rRNA was amplified through PCR using specific primer (515F: CCTAYGGGRBGCASCAG; 806R: GGACTACNNGGGTATCTAAT) with the barcode, then purified with GeneJETTM Gel Extraction Kit (Thermo Scientific). Sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns (Thermo Scientific) following manufacturer’s recommendations. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific). The library was sequenced on an Ion S5TM XL platform and 400 bp/600 bp single-end reads were generated. Sequences analysis were performed by Uparse software (Uparse v7.0.1001), and the analysis of diversity were calculated with QIIME (Version1.7.0) and displayed with R software (Version 2.15.3). The concentrations of SCFAs, including acetic acid, propionic acid and butyric acid, were detected on gas chromatography (GC) after several procedures for pretreatment based on the method of previous study (Fu et al., 2018a). They were quantitated according to the standard curve with crotonic acid as internal standard.
Statistical Analysis
The SPSS 22.0 software was used to carry out a One-way ANOVA test, followed by LSD post-hoc test. All the experimental data were expressed as Mean ± SD (‾x ± SD) in table and Mean ± SEM (‾x ± SEM) in figures. The p-value of 0.05 or less was considered with statistically significance.
Discussion
As a common medicinal material, C. pilosula and its polysaccharide component have presented versatile pharmacological activities. Particularly, the polysaccharide from C. pilosula have been evidenced possessing both prebiotic and immunomodulatory activities in vitro or in vivo, by a range of previous studies (Peng et al., 2018; Deng et al., 2019) and experimental results in our group, including crude polysaccharide CPP (Fu et al., 2018a), inulin-type fructan CPPF (Fu et al., 2018b) and pectic polysaccharides (Zou et al., 2019). Considering its macromolecular and non-digestible properties in upper gastrointestinal tract, such natural polysaccharides can reach to intestine without degradation because of the limited hydrolysis enzymes in human (Wilson and Whelan, 2017; García Gamboa et al., 2018). Considering the modulation effects of Codonopsis-derived polysaccharides (Zhou et al., 2015; Li et al., 2018; Zou et al., 2019) or inulin (Vogt et al., 2015; Akram et al., 2019) on intestinal mucosal immune, the intestinal mucosa and/or gut microbiota were considered as a potential way that CPPF probably act.
Studies have demonstrated that CY destroys intestinal mucosal system by reducing barrier function, injuring intestinal villi and crypts, inhibiting secretion of sIgA and damaging Peyer’s patch (Miao et al., 2015; Zhao et al., 2016; Wang Z et al., 2019). In gastrointestinal mucosal barrier, goblet cells continually produce mucins to replenish and maintain the mucus barrier, where some chemical composition like sIgA is higher concentrated (Ouwerkerk et al., 2013; Cornick et al., 2015). The Muc2 and sIgA secretion were facilitated by CPPF, in accordance with other inulin reported before (Ito et al., 2008; Nagata et al., 2018). It could be directly resulted from activation of mucosal receptors cooperated with downstream signal pathways, or gut-associated lymphoid tissues (GALTs). Secretion cells, such as goblet cell, Paneth cells and enteroendocrine cells could also be involved (Kim and Ho, 2010; Vogt et al., 2015). It also possibly came from the intestinal bacteria change in an indirect way (Kim and Ho, 2010), that the abundance of certain mucus-degrading bacteria could be reduced (Schroeder et al., 2018; van den Abbeele et al., 2018). Thus, detailed composition changes in gut microbiota were analyzed.
Tissues with high proliferation rate, like intestinal epithelia cells, are vulnerable to CY because of its ability of killing dividing cells. Those damaged cells and produced reactive oxygen species (ROS) will induce apoptosis and up-regulate inflammatory cytokines (Taminiau et al., 1980; Moore, 1984; Sonis, 2004). There are numerous studies indicated that the treatment of CY could promote inflammatory cytokine secretion like IL-1β, TNF-α of porcine epithelial cells (Lee and Kang, 2017) or in small intestine (Shi et al., 2017; Wang X et al., 2019). Moreover, commercial inulin from Mexican blue agave (Agave tequilana Weber var. azul) or Chicory have been used as nutritional supplements with an anti-inflammatory effect in intestine (Rivera-Huerta et al., 2017; Akram et al., 2019; Zhao et al., 2019). However, it was very likely the first time showing the active target location of inulin from natural medicinal plants. Some studies indeed found an anti-inflammatory effect in intestinal mucosa of polysaccharide from C. pilosula (Zhou et al., 2015; Chen, 2016; Li et al., 2018). But no comprehensive investigation was declared that which intestinal part it can work on. Combing with previous studies of LCP-II-I (Huang C et al., 2017), we now have sufficient experimental evidence proving that these polymers could be active only before colon in vivo, since they could already been degraded or fermented in colon. In addition, the main factor of fermentation rate of inulin could be the DP variation (Gibson and Delzenne, 2008). Zhao et al. (2019) revealed that 28 days of short chain fructooligosaccahrides (scFOS) with maximum DP of 6, could down-regulate IL-1β expression in jejunal mucosa, but not active in ileum. Comparatively, the DP of CPPF in our study was higher (about 2–17) (Fu et al., 2018b), and could be degraded much slowly and even far in intestine tract. That was probably the main reason that CPPF displayed well on anti-inflammatory effect in both jejunum and ileum.
Polysaccharides from marine animals, fruits, vegetables and natural medical plants, have been found with high fermentable and modulatory effect by gut microbiota (Bosscher et al., 2006; Xu et al., 2013; Shoaib et al., 2016; Shang Q et al., 2018). Although immunosuppressed mice were treated with CPPF for only 7 days, there was still bacterial community change observed in our research. Some similar studies (Chen et al., 2017; Moens and De Vuyst, 2017; Zhu et al., 2017; Hoving et al., 2018; Baxter et al., 2019; Li et al., 2019) also manifested that the abundance of Clostridium, Deferribacteres, Ruminococcaceae, Lachnospiraceae or Oscillospira were regulated by several weeks of inulin treatment. As one of probiotics, Ruminococcceae has been well reported with a responsible relation to polysaccharides degradation, like fucoidans and inulin (Shang et al., 2016; Moens and De Vuyst, 2017). In this study, a remarkable increase of unidentified Ruminococcaceae was demonstrated by CPPF, which proved it with a prebiotic activity. CPPF also displayed an ameliorated effect by decreasing the amount of Mucispirillum schaedleri, which is parasitic in intestinal mucous layer, and can easily translocate in intestinal mucosa and activate NF-kB or PPAR-delta receptors to trigger inflammatory response (Loy et al., 2017). The Oscillospira recovered by CPPF is one of the butyrate-producing bacteria, and are usually considered as protector against several pathogens by acidifying intestinal environment. This recovery has also been achieved by polysaccharides from purple sweet potato on immunosuppressed mice (Tang et al., 2018). Moreover, it was found that the Blautia in the family of Lachnospiraceae was promoted after CPPF administration as shown in Figure 5D. It has been proved that the relative abundance of Lachnospiraceae has significant correlation with host physiological dysfunctions, such as obesity, diabetes, cancer, and various inflammatory diseases, and could be an underlying probiotic (Liu et al., 2021).
Furthermore, the relative abundance of Lactobacillus was displayed an opposite trend with our previous results, that it was reduced relatively in mice treated with inulin, which was also showed in study of Hoving et al. (2018). After reviewing the overall microflora data, we inferred that it might be due to the different Lactobacillus species that we used in previous and current study. And the overwhelming reduction by CY on the abundance of other bacteria could lead to a relatively increase of Lactobacillus. A comparatively lower abundance in CPPF groups may therefore be observed. The dose could be another factor leading to a lower prebiotic effect. No significant change on the relative abundance of Bifidobacteriaceae and Lactobacillus was observed after even above 1.25 g/kg of inulin/FOS administration (Zhu et al., 2017).
As one of the non-digested polymers, inulin can reach to intestine intactly, and serve as energy source for beneficial bacteria that generate metabolites like SCFAs. The increase of butyric acid, the main one utilized by colonocytes, was related to a higher abundance of genera Ruminococcaceae (Biddle et al., 2013; Fernández et al., 2016), which produces butyrate as most abundant family in the order Clostridiales. Additionally, Ruminococcaceae was indicated with positive correlation with butyrate/SCFA ratio in vivo (Biddle et al., 2013). And the Oscillibacter, Ruminococcus and Papillibacter, belonging to family Ruminococcaceae, were all promoted by CPPF in our study. This alteration on bacterial composition could be the main reason of the certain high butyric acid in CPPF group compared with CY group, similar with previous studies (Moens and De Vuyst, 2017; Baxter et al., 2019). The acetic acid is produced by most enteric bacteria. Its dramatically drop in cecal content could because of the lower bacterial OTUs induced by CY. While the less restoration in CPPF groups could be due to the lower abundance of acetic acid-producing bacteria, like Lactobacillus (Fernández et al., 2016) (Figure 5). The relative abundance of genera Bacteroides, who converts sugars to beneficial metabolites, like propionic acids (Biddle et al., 2013; Fernández et al., 2016), were higher in CPPF-H group than CY group, and was coincident with results of SCFAs levels. Propionic acid could be produced mainly by ketone fermentation (e.g., fructose, arabinose and tagatose) in vitro (Hu et al., 2013). The abundant fructose in CPPF could be one of the reasons of its higher concentration. Comparatively, the acetic and butyric acids are produced mainly by aldehydes fermentation (e.g., glucose, galactose, mannose and xylose) (Hu et al., 2013). These changes on acetic and butyric acids are likely coming from bacteria modulated by CPPF in current research, rather than its degradation metabolites, as CPPF is consisted of a lower amount of glucose relatively to fructose. However, such advantageous SCFAs were not promoted by CPPF. The possible reason is that the mice were not intervened enough to show a significantly change on gut flora and carbohydrate fermentation. And the relative abundance of those SCFAs-producing bacteria could be influenced by complicated micro-environment, which is also a challenge of studies on microbiota and polysaccharide degradation.