Zhihong HUANG

College of Life Science and Technology, Southwest Minzu University, Chengdu 610041, China

Abstract [Objectives] This study aimed to investigate the effect of polysaccharides from Hedyotis diffusa on the immune cells of the intestinal mucosa of chicks. [Methods] Total 200 seven-day-old AA chicks were evenly and randomly divided into four groups: control group and three treatment groups. The chicks in the control group (group I) were fed basal diet, and those in the treatment groups were fed with basal diet supplemented with 0.4 (group II), 0.8 (group III) and 1.2 (group IV) g/kg H. diffusa polysaccharides (OPS), respectively. At the age of 14, 28 and 42 d, 10 chicks from each of the groups were sacrificed, respectively for counting of intraepithelial lymphocytes (IELs) and lamina propria secretory IgA (SIgA) positive cells in small intestinal mucosa. [Results] At the age of 14 d, the duodenal and jejunal IEL and SIgA positive cell counts in groups II, III and IV were significantly greater than those in group I (P<0.05). At the age of 28 d, the jejunal IEL count of group IV was significantly lower than those of groups I, II and III (P<0.05); and there was no significant difference in IEL or SIgA positive cell count between group I and the other groups (P>0.05). At the age of 42 d, the duodenal and jejunal IEL counts in group II were significantly greater than those in groups I, III and IV (P<0.05), while the differences in duodenal and jejunal IEL count between groups III, IV and I were insignificant (P>0.05). The duodenal SIgA positive cell counts in the treatment groups were insignificantly different from that in group I (P>0.05). The jejunal SIgA positive cell count in group II was significantly greater than those in groups I, III and IV (P<0.05). There was no significant difference in jejunal SIgA positive cell count between groups III and IV and group I (P>0.05). [Conclusions] OPS at 0.4 g/kg can promote the proliferation of immune cells in the small intestine mucosa of 14-42-day-old AA chicks, and it is recommended to be added to the feed.

Key words Hedyotis diffusa, Polysaccharide, Intraepithelial lymphocyte, SIgA positive cell, Duodenum, Jejunum

1 Introduction

2 Materials and methods

2.1 Materials

2.1.1Experimental drug. OPS (45% content) was provided by Beijing Centre Technology Co., Ltd.

2.1.2Experimental animals. A total of 200 healthy one-day-old AA chicks, half male and half female were used. They were purchased from Chengdu Zhengda Co., Ltd.

2.1.3Main instruments and reagents. Paraffin embedding machine (LEICAEG1150) and rotary slicer (LEICARM2245) were produced by Leica Biosystems (Germany). Motic optical microscope (B5 professional Series) was produced by Bock Optronics Incorporation (Canada). Olympus microscope (CX21FS1) was produced by Olympus Corporation (Japan).

Goat anti-chicken IgA monoclonal antibody was provided by Shanghai Baili Biotechnology Co., Ltd. SP-9000 kit (including endogenous peroxidase blocker, normal goat serum working solution for blocking, universal biotin-labeled secondary antibody working solution and streptavidin-horseradish peroxidase working solution) was provided by ZYMED Laboratories Incorporation (America).

2.2 Methods

2.2.1Animal grouping and sample collection. After one week of adaptive feeding, the 200 seven-day-old chicks were randomly divided into a control group and three treatment groups, with 50 animals in each group, half male and half female. The chicks in the control group (group I) were fed basal diet, and those in the three treatment groups were fed basal diet supplemented with 0.4 (group II), 0.8 (group III) and 1.2 (group IV) g/kg OPS, respectively, for one week. On days 14, 28 and 42, 10 chicks were selected randomly from each group and sacrificed for collection of duodenal and jejunal tissues, which were fixed with paraformaldehyde.

2.2.2Counting of IELs and SIgA positive cells in intestinal villi. Paraffin sections, 4 μm thick, were sliced by conventional method, stained with hematoxylin-eosin (HE) or immunohistochemical kit (operate according to the kit instructions), and observed and photographed with an optical microscope. For each section, 10 longest, straight and well-stretched villi were selected. They were analyzed quantitatively using Motic photographic processing software and pathological image analysis system. The number of intraepithelial lymphocytes (IELs) in the mucosa and the number of SIgA positive in the lamina propria per unit length (1 mm) of intestinal villi were counted.

2.2.3Statistical analysis. The experimental data were subjected to one-way analysis of variance using SPSS 18.0 software, and the analysis results are expressed as mean±standard deviation. The significance of difference between the data was analyzed through the homogeneity test of variance. WhenP<0.05, the difference is considered significant.

3 Results and analysis

3.1 SIgA positive cell countIn the SIgA-positive cells, the nuclei were stained blue, and the cytoplasm was stained brown (Fig.1).

Fig.1 SIgA positive cells in jejunum (IHA, 400×)

3.1.1Duodenum. On day 14, the duodenal SIgA positive cell counts in groups II, III and IV increased significantly compared with that in group I (P<0.05), while no significant difference was observed between groups II, III and IV (P>0.05). On days 28 and 42, there was no significant difference between the groups (P>0.05) (Table 1). Among the groups, the effect of group II was better.

Table 1 Count of SIgA positive cells in duodenum

3.1.2Jejunum. On day 14, the counts of SIgA positive cells in groups II, III and IV were all greater than that in group I (P<0.05), while there was no significant difference between groups II, III and IV (P>0.05). On day 28, no significant difference in SIgA positive cell count was observed between groups I, II, III and IV (P>0.05). On day 42, the SIgA positive cell count of group II was significantly greater than those of groups I, III and IV (P<0.05), while there was no significant difference among groups I, III and IV (P>0.05) (Table 2). In summary, the effect of group II was better.

Table 2 Count of SIgA positive cells in jejunum

3.2 IEL countIELs are located between the intestinal villous epithelial cells, mostly near the basement membrane. The cells are medium in size, with deep stained nuclei and less cytoplasm. The cytoplasm is stained darker than the adjacent intestinal epithelial cells (Fig.2).

Fig.2 Intraepithelial lymphocytes (IELs) in jejunum (HE, 400×)

3.2.1Duodenum. On day 14, the duodenal IEL counts of groups II, III and IV were significantly greater than that of group I (P<0.05), while there was no significant difference between groups II, III and IV (P>0.05). On day 28, no significant difference was found in duodenal IEL count among the groups (P>0.05). On day 42, the duodenal IEL count of group II was significantly greater than that of group I (P<0.05), and the differences between groups III and IV and group I were insignificant (P>0.05) (Table 3). In summary, the effect of group II was better.

3.2.2Jejunum. On day 14, the jejunal IEL counts of groups II, III and IV were significantly greater than that of group I (P<0.05), and the differences between groups II, III and IV were insignificant (P>0.05). On day 28, the jejunal IEL count of group IV was significantly lower than those of the other groups (P<0.05), and there was no significant difference between groups I, II and III (P>0.05). On day 42, the jejunal IEL count of group II was significantly greater than those of groups I, II and IV (P<0.05), and there was no significant difference between groups I, III and IV (P>0.05) (Table 4). In terms of effect, group II was better.

Table 4 Count of IELs in jejunum

4 Discussion

The digestive tract mucosa is the body’s first line of defense against the invasion of foreign pathogens. Intraepithelial lymphocytes are one of the main active cells of the intestinal mucosal immune system. When the body is affected by external stimuli or pathogens, the imbalance between quantity and function may cause a variety of intestinal or autoimmune diseases. SIgA produced by SIgA-positive cells is the main effector molecule of the mucosal immune system. It can prevent microorganisms from breaking through the intestinal mucosal barrier by binding antigenic substances in mucus.

4.1 Effect of OPS on the number of SIgA positive cells in the small intestineA variety of polysaccharides can increase the content of SIgA in the intestine. At 14 d of age, OPS can promote the proliferation of SIgA positive cells in the duodenum. The research results of Zuo Taoetal.[8]show that squid ink polysaccharide can promote the secretion of SIgA in mouse intestinal mucosa. Wang Qingetal.[9]found thatPoriapolysaccharides can promote the production of SIgA in the intestine of mice. Shan Chunlanetal.[10]showed thatAstragaluspolysaccharides can significantly increase the IgA+cells in the duodenum of chicks. In the study conducted by Zhang Dapengetal[11], it was found that total polysaccharides of Sijunzi Decoction can increase the level of SIgA in the intestinal tract of immunosuppressed mice. OPS may have similar effects as squid ink polysaccharides,Poriapolysaccharides,Astragaluspolysaccharides and Sijunzi Decoction total polysaccharides to promote intestinal IgA secretion.

4.2 Effect of OPS on the number of IELs in the small intestineMany factors can change the proliferation level of small intestinal IELs. OPS can increase the counts of duodenal and jejunal IELs in AA chickens. The proliferation effect of IELs in each part of the small intestine is most significant at 14 d of age. OPS promotes the proliferation of IELs in the small intestine and is similar to the effects ofAstragaluspolysaccharides, sulfatedAstragaluspolysaccharides, andAntenoronfiliformepolysaccharides[12-13].

OPS has anti-oxidation, immune regulation, anti-tumor and other biological activities, and the mechanism of action is related to improving immunity and scavenging free radicals[14]. Jiang Jianpingetal.[15]conducted aninvitroantioxidant activity test of OPS. The results show that OPS at a concentration of 1 mg/mL has strong antioxidant capacity, and the scavenging rate of DPPH free radical is as high as 82.66%. The research results of Cai Wei[16]show that OPS powder can significantly improve the level of cellular immunity and humoral immunity in mice, and significantly improve the phagocytic capacity of mouse peritoneal macrophages and the organ index of the spleen. At the same time, it also significantly promotes the weight gain of mice. Whether OPS promotes the proliferation of small intestinal mucosal immune cells is related to its immune enhancement and antioxidant effects remains to be further studied.

During the raising of chicks, especially broilers, the morbidity and mortality of colibacillosis, salmonellosis, Newcastle disease, bursal disease, infectious anemia, coccidiosis,etc. remain high. This is also an important reason for slower weight gain and lower feed efficiency for broilers. Many diseases such as bursal disease and infectious anemia can also cause immunosuppression. As a result, the immune effect of broiler vaccines is often not ideal. Therefore, it is of great significance to explore the use of plant polysaccharides with immune regulation and growth-promoting effects as feed additives for broilers. The results of this test show that OPS can promote the proliferation of IELs and SIgA positive cells in AA chickens. The proliferation effect is strongest at 14 d of age. Therefore, adding OPS to the early feed of broilers may help resist the invasion of microorganisms and reduce the morbidity and mortality of broilers, thereby improving the production performance of broilers.

5 Conclusions

OPS can promote the proliferation of small intestinal mucosal immune cells and improve the immune function of small intestinal mucosa of chicks. It has a better effect on young chicks. Therefore, OPS can be added to the starter feed of broilers, and the addition amount is preferably 0.4 g/kg.