Protective Effects of Atractylodes macrocephala Polysaccharide on iIELs and the IL-6, TNF-α mRNA Level in E. coli induced Diarrheal Mice

Zhongqing Wang1, Qiuyue Wu1, Ruiling Ye1, Fazul Nabi1,4, Yangfei Shang1, Sarfaraz Ali4 and Juan Liu1,2,3* 1College of Veterinary Medicine, Southwest University, Rongchang, Chongqing 402460, P.R China 2Chinese of Veterinary Drug Innovation R and D Lab of Chongqing Engineering Research Center of Veterinary Science, Rongchang, Chongqing 402460, P.R. China. 3Immunology Research Center, Southwest University Medical Research Institute, Rongchang, Chongqing. 402460, P R China. 4Faculty of Veterinary and Animal Sciences, LUAWMS, Uthal, Pakistan.


INTRODUCTION
D iarrhea is a common problem with public health and animals, one of the leading causes of antibioticassociated and widespread around the world (Alebel et al., 2018;Chang et al., 2018) results in high morbidity and mortality. Many pathogens and several emerging species threat the animal production and losses the livestock mainly in developing countries (Dunn and Johnson, 2018). Enterotoxigenic Escherichia coli is well-known pathogen that cause diarrhea. Specifically, it adheres to the O n l i n e

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and represent one of the single largest populations of lymphocytes in the body (Van Kaer and Olivares-Villagómez, 2018). iIELs could be divided into type a or conventional T cells and type b or nonconventional T cells based on T-cell receptor (TCR) usage and expression of coreceptors (Taveirne et al., 2011). CD4+T cells in iIELs could attenuate the adjusting of inflammation and immunity, which enhanced the effect of self-protection (Cheroutre, 2005). Furthermore, report indicated that the diarrhea induced by LPS (the main structure of E. coli) could be cured by plants polysaccharides as well (Sousa et al., 2016). LPS-induced tissue damage related to NF-κB inflammatory signaling pathway which prevailingly mediated by TNF receptor associative factors, such as IL-6, IL-8 and TNF-α. A. macrocephala (Fig. 1A) is a traditional Chinese medicine that derived from the rhizoma of A.macrocephala Koidz., used to treat gastrointestinal ulcers, lower immunity, chronic wasting disease, cancers and diarrhea (Guan et al., 2018;Huang et al., 2005;Zhang et al., 2017). A. macrocephala mainly contains polysaccharide, lactone, volatile oil and sesquiterpenes (Chou et al., 2012;Kang et al., 2011), which is used as an anti-bacteria, antiinflammation and anti-tumor properties (Dong et al., 2008;Zhang et al., 2000). In our previous research, we confirmed that the extract of zhuqin compounds enhanced the immunity of immunosuppressed in mice (Juan et al., 2015). Polysaccharides as the vital material basis for Chinese herbal medicine perform unique therapeutic effects such as regulating immunity, apoptosis and antitumor mediator Wei et al., 2018;Li et al., 2018;Fan et al., 2017;Xu et al., 2014). Previously we found that the total polysaccharides of Scutellaria and A. macrocephala (including AMP) maintained the integrity of immune function via inhibiting the autophagy of spleen lymphocytes in mice (Pang-min, 2018). Therefore, the aim of this study was to investigated the effects of AMP on E. coli induced diarrhea on the ability to affect the quantity of iIELs in small intestine and the mRNA transcription of TNF-α and IL-6 in duodenum of mice.

Preparation and identification of AMP
AMP was prepared according to the previous study (Pang-min, 2018). AMP was primitively identified by the tests of molish, biuret reaction, Fehling reagent reaction and iodine-potassium iodide reaction. Phenol-sulphate acid method (Zhao, 2005) was adapted to measure the purity of AMP. The average absorbance (AA) of 2, 4, 8, 16, 32, 64 mg/ml glucose solution were collected, and then the standard curve was drawn. The AA of AMP was measured and the polysaccharides concentration was calculated by the standard curve, and the purity (m%) followed the formula: m%= 100×m 1 /m 2 [m 1 : polysaccharides concentration; m 2 : AMP concentration (50.4 mg/L)].

The structure of AMP analysis by fourier transform infrared spectrometry (FTIR)
A small amount of AMP (3 mg) were weighed and added to KBr, then crushed into thin slices. FTIR technique was used to scan infrared wave number in the range of 4 000 cm -1 -400 cm -1 , and the height ratio of characteristic peak was calculated and analyzed by FTIR.

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Animals and treatment A total 72 Kunming strain mice (20±2 g, male and female in equal), were purchased from the experimental animal, Center College of Animal Science, Southwest University Chongqing China. After one-week adaptive feeding, mice were randomly divided into 6 groups equally (n=12). The positive drug group (PG) was administered with 1.2 mg/mL astragalus polysaccharide orally, while the AMP low, medium and high dose groups (LG, MG, HG) were administered with 1.2, 3.6, and 6 mg/mL of AMP, respectively. The control group (CG) and infection group (IG) were administered with equal volume of normal saline. Mice were administered with E. coli 0.5 mL/time/ day for 7 days. All experiments were. All animal research experiments were conducted according to the National Institutes of Health guidelines for experimental animal use and were approved by the Institutional Animal Care and Use Committee at the Southwest University, Rongchang, Chongqing China. Mice were housed at standard conditions (23±2 °C; 60% humidity; 12 h light-dark cycle) and fed ad libitum standard diet during the whole experiment period. After the last administration subsequent 24 h, mice in each group were intraperitoneally (i.p) injected with E. coli (5×10 7 CFU) to induce diarrhea except CG.

Clinical observation and management
During the clinical observations when mice show the depressed spirit, decreased appetite and laxly in IG the point time was set as 0 h. The incidence of diarrhea was calculated at 6 h. To evaluated the protective effect of the drugs on diarrheal mice, we set fecal consistency of scores in mice according to the previous method (0, normal; 1, soft faeces; 2, mild diarrhea; and 3, severe diarrhea) (Liu et al., 2016). The fecal consistency of scores in mice were collected at 4 d by a trained individual, but who had no prior knowledge of treatment each mouse had received.

The quantity of iIELs in small intestine analyzed by moditec
At 6 h and 4 d, 6 mice from each group were euthanized by cervical dislocation. Intestinal segment (duodenum, jejunum, and ileum) samples were fixed in 10% Formalin solution, embedded paraffin, sectioned, stained with H and E staining (Estrada et al., 2005). The quantity of iIELs between 100 columnar epithelial cells in intestinal mucosa was counted by Moditec photo processing software under the light microscope (Jin-kun et al., 2008). Three distinct regions of each intestinal segment were selected as average value for iIELs analysis.

IL-6 and TNF-α mRNA expression level in duodenum
At 6 h and 4 d, the duodenal segments from 6 mice from CG, IG, PG and HG were collected. Duodenal segments were rinsed with PBS buffer to clean the intestinal contents, put into the EP tube (without RNA enzyme), and stored at -80 o C for further analysis. The primers selected for the current study was designed by GenBank, (Table  I). Duodenal tissue samples (50mg) were collected for the synthesis of RNA and cDNA. RNA was extracted by B511321-UNIQ-10 total RNA extraction kit, and reverse transcribed into cDNA by primer-script TM reagent kit. qRT-PCR reaction was performed briefly a total reaction volume system (20 μL) contains 2 × SYBR premix ex Taq mix 10 μL, QF (10 μmol/L), QR (10 μmol/L) respectively 0.5 μ L, the template cDNA 2 μL, and 7 μL RNase free dH 2 O. The amplification procedure parameters contain the 94 o C for 30 s, 94 o C 5 s, 61 o C 35 s, with 40 cycles, then the specificity of products were detected by dissolution curve. Dissolution parameter contains the: 97 o C for 10 s, 65 o C 60 s, 97 o C 1 s, 1 cycle. The ΔCt value of the amplification each curve of cDNA sample was recorded, and the relative level of mRNA expression was calculated by 2 -ΔΔCt method (Ghowsi et al., 2018).

Statistical analysis
The data were expressed by mean ± standard deviation, and analyzed by SPSS 20.0 statistical software, P < 0.05 was the significant difference, P < 0.01 was the highly significant difference.

Identification of AMP
As shown in Figure 1, The polysaccharides concentration (49.454 5 mg/L) was calculated, and the purity (m%) of AMP was 98.12%. The results illustrated that AMP contained polysaccharide, instead of proteins, reducing sugars and starch. As shown in Figure 2, the strong vibrations arose in characteristic spectral band of 3 446.61 cm -1 and 3 351.41 cm -1 indicated that the stretching vibration absorption peaks of -OH. The characteristic spectral band of 2 934.88 cm -1 and 1 637.69 cm -1 severally signified absorption peak of -CH 2 and -COOH. The -C-H vibration absorption peak at 1 402.64 cm -1 , suggested that AMP included ring structure with carbon atom. The spectral band of 1 131.90 cm -1 , 1 036.69 cm -1 and 941.49 cm -1 were the characteristic absorption peaks of pyranose. The spectral band of 816.53 cm -1 was absorption peak of alpha-glycosidic bond. The results implied that AMP might be a class of pyran polysaccharide with alphaglycosidic bond.

Protective effect of AMP on diarrheal mice
During the whole period of treatment the mental state, drinking, appetite and physical activity of mice in

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each group was normal with no signs of diarrhea. At 6 h observation, the mental state and appetite of mice in CG was normal. Conversely, the mice showed the abnormal mental state and the lack of the appetite in IG. Although, the abnormal mental state and appetite of mice in PG, LG, MG and HG group's shows nearly to the normal levels. The incidence and protective rate of diarrhea in each group is shown in Table II. The fecal score of IG was significantly increased (P<0.01) as compare to CG. As compared with IG, the faecal score of PG, LG, MG and HG groups were decreased significantly by dose-dependently decreased (P<0.01) ( Table II).

Protective effect of AMP on the intestinal histology
Duodenal mucosa in CG was clear polarized, the morphology of intestinal epithelial cells was normal, the nucleus with dark blue was localized at the base of the enterocyte and the cytoplasm was seems uniformly red stained. The duodenal mucosa in IG was seriously injured; the intestinal epithelial cells show hyperplasia, flattened crypts, pyknosis and necrosis. However, PG, LG, MG and HG groups' intestinal histology were improved and duodenal mucosa with normal structure of villi and crypts, and blue stained nuclei and normal morphology (Fig. 3).

Quantity of iIELs in small intestine of E. coli induced diarrheal mice
At 6 h, the quantity of iIELs in small intestine of IG was significantly higher than that in CG (P<0.01); As compared with IG, the quantity of iIELs in small intestine of PG, MG and HG groups was significantly decreased (P<0.01), and that in LG group was decreased (P<0.05 or P>0.05). At 4 d, the quantity of iIELs in duodenum and ileum of IG was significantly higher than that in CG(P<0.01); As compared with IG, the quantity of iIELs in duodenum and ileum of PG and HG groups were significantly reduced (P<0.01), and that in MG and LG groups were reduced (P<0.05 or P>0.05). However, at 4 d, the quantity of iIELs in jejunum of IG significantly lower than that in CG (P<0.01). The quantity of iIELs in jejunum of PG, LG, MG and HG groups were significantly increased as compare with IG (P<0.01) (Fig. 4).    Expression levels of IL-6 and TNF-α in duodenum of mice The mRNA level of IL-6 in duodenum of IG was significantly increased at 6 h and 4 d in comparison to CG respectively (P<0.01). However as compared with IG, the mRNA level of IL-6 in duodenum of PG and HG gruops was decreased significantly (P<0.01). The mRNA level of TNF-α in duodenum of IG was significantly increased at 6 h and 4 d as compared to CG (P<0.01). Although the mRNA level of TNF-α in duodenum of PG and HG groups decreased significantly as compared with IG (P<0.01) (Fig. 5).

DISCUSSION
Intestinal intraepithelial lymphocytes iIELs are various populations of lymphoid cells that inhabit between the intestinal epithelial cells to form the intestinal mucosal barrier. The intestinal epithelial cells and iIELs are important components of the intestinal immune system against invading pathogen, they interface for immune cells to detect and respond to environmental substances (Suzuki, 2012). It is found that the excessive iIELs could reduce the functions of intestinal mucosal barrier (Hayday et al., 2001). However, the quantity of iIELs might be relative to the contact with the microbiota and pathogen types and dose of invading pathogen, also the functions of innate immune and mucosal immune response. The increase of iIELs was a characteristic index for early diagnosis of gastrointestinal diseases and tissues damages, which might be related to peptic duodenitis (Chang et al., 2005). Our findings revealed that the quantity of iIELs in O n l i n e

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Z. Wang et al. small intestine of diarrheal mice was increased with E. coli induction suggested that E. coli might induce extensive enteritis in small intestine of the mice. The amount of iIELs in small intestine of diarrheal mice was decreased in dosedependent manner with the pre-administration of AMP, which indicated that AMP could maintain the functions of intestinal mucosal barrier and as an anti-inflammation. The epithelial cells intestine proliferated rapidly to maintaining the steady state of intestinal mucosa, therefore the number of iIELs in diarrheal mice was relatively decreased. The pre-administration of AMP promoted the increase of iIELs in jejunum of diarrheal mice, which manifested that AMP maintained the functions of intestinal mucosal barrier by facilitating the proliferation of intestinal epithelial cells.
The AMP regulates the disorder of intestinal flora, and improve the metabolism of animals (Wang et al., 2014). The effect of AMP on anti-pathogenic microorganism found better than antibiotics (Li et al., 2011), and it could promote lymphocyte proliferation after selenium modification (Liu et al., 2015). AMP possess biological activities to improve in digestive system, anti-tumor, antiinflammatory, anti-aging, anti-oxidative, and anti-bacterial activities (Li et al., 2012Shu et al., 2017;Wang et al., 2009Wang et al., , 2014Kim et al., 2016). IL-6 is considered a mediator of the inflammatory response and it has many biological functions induced by iIELs, which can protect the colon from invasion by pathogen and the integrity of intestinal mucosa (Kuhn et al., 2018). IL-6 acts on pro-inflammation, stimulates the inflammatory injury and the development of tumors when largely proliferation (Fizazi et al., 2012). The iIELs of TCRβ + CD4 − CD8 − in colon of mice could produce a large number of IL-6 in the early stage of inflammatory injury (Kuhn et al., 2014). The present study showed that the expression of IL-6 in the duodenum of diarrheal mice was significantly higher than those normal mice. The preadministration of AMP decreased the mRNA transcription of IL-6 in the duodenum of diarrheal mice; our results were consistent with other previous research (Frech and Hudson, 2015;Herlina et al., 2016;Pei et al., 2019;Zhou et al., 2006) as well. The study suggested that AMP effectively antagonize duodenitis and diarrhea in mice and protective effects in intestinal cells and tissues.
The production of inflammatory mediators is cascade process, and the NF-κB signaling pathway is a classical pathway that induces inflammatory mediators. The study found that TNF-α could be involved in the activation of NF-κB inflammatory signaling pathway, which ultimately activates the inflammatory cells to express more inflammatory factors such as IL-6, TNF-α, etc. (Kuhn et al., 2018). Many diseases are associated with excessive secretion of inflammatory mediators (Tsoulfas and Geller, 2001). The anti-TNF-α therapy could effectively cure rheumatoid arthritis and prove that TNF-α playing an important role in the development of inflammation (Feldmann and Maini, 2001). TNF-α highly expressed due to the stimulating ROS production in intestinal epithelial cell (Rokutan et al., 2008), which could induce the chronic inflammatory bowel disease (Ahn and Kim, 2018). Our study shows that the transcription of TNF-α in duodenum of diarrhea mice was increased, suggested that the inflammatory response of duodenum in diarrhea mice might relate to the activation of NF-κB inflammatory signaling pathway. However, the pre-administration of AMP reduced transcription level of TNF-α in diarrhea mice, which was consistent with the previous results (Belluzzo et al., 2014;Park et al., 2017;Yang et al., 2005). The anti-inflammatory mechanism might be related to the inhibition of NF-κB inflammatory signaling pathway activation.

CONCLUSION
In conclusion, the results reported here show that AMP is a potential alternative approach for treating diarrhea condition by regulating the quantity of iIELs in small intestine. The anti-inflammatory effect of AMP is associated with the down-regulation of the IL-6, TNF-α transcriptional level and the balanced pro-inflammatory cytokine in diarrheal mice.

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Effects of Atractylodes macrocephala Polysaccharide in Diarrheal Mice