Effects of Traditional Chinese Medicine on Numbers of Lymphocytes and Goblet Cells in Villus Epithelia of Layers under Heat Stress

Qiumei SHI,Guisheng GAO,Chao XING,Huan LIU,Ping SHEN,Fangfang PAN,Guangping GAO,Yanying ZHANG

Hebei Province Key Laboratory of Preventive Veterinary Medicine,Hebei Normal University of Science and Technology,Qinhuangdao 066004,China

Responsible editor:Tingting XU Responsible proofreader:Xiaoyan WU

The metabolism inside body will change with the change in environmental temperature.So an appropriate environmental temperature can therefore maintain the metabolic strength and heat production inside body at the lowest levels in physiology.This temperature range is defined as zone of thermal neutrality or thermal neutral zone.The thermal neutral zone is most favorable for livestock.This is because that an excessively low temperature will lead to the increase of feed consumption,and an excessively high temperature will reduce the performance of animal.For chickens,the thermal neutral zone is 15-16 ℃.The thermal neutral zone of broilers is 10-27 ℃,and of layers is 13-25 ℃.When the environmental temperature is above the thermal neutral zone,the specific responses will occur in chicken.This phenomenon is also called as heat stress[1].

Intraepithelial lymphocytes (IELs)are widely distributed in the epithelial tissue of skin,respiratory tract,gastrointestine and genitourinary tract.The intraepithelial lymphocytes that are distributed in the epithelia of gastrointestine are also called as intestinal intraepithelial lymphocytes (IELs).Since the intestinal intraepithelial lymphocytes are long-termly contacted with intestinal flora and pathogens,they have essential immune function for bodies[2].

Goblet cells are scattered among columnar cells.The secretion of goblet cells is rich in mucin.The mucin is mixed with water on the surface of intestinal epithelia,forming into the mucus barrier,which plays a mechanical protecting effect on intestinal mucosa[3].In addition,the mucus barrier can also lubricate the intestinal epithelia.In addition to mucin,the goblet cells also secret a substance called as trefoil peptide,which is combined with mucin-like glycoproteins,forming into a layer of continuous gel on the mucosal surface.The combination can protect the intestinal mucosa from chemical and biological damage[4].

Chinese herbal medicine is rich in vitamins,trace elements and other active ingredients.It can enhance the immunity and comprehensively coordinate physiological metabolism,thereby relieving heat stress.Studies have shown that the anti-heat stress formula,consisting of gypsum,Radix isatidis,Scutellaria baicalensis,Atractylodes lancea,Radix paeoniae Alba,Codonopsis pilosula,Lophatherum gracile,Glycyrrhiza uralensis Fisch.,can significantly improve the yield and quality of milk and the immunity of cows in summer[5].Another study showed that,under conditions of 38 ℃of temperature,80% of humidity and 1 h of exposure to sunlight,the oral administration of Codonopsis pilosula,Schisandra chinensis,Scutellaria baicalensis,Glycyrrhiza uralensis Fisch.and Lespedeza bicolor could significantly enhance the thermal tolerance in rats,reduce heat damage to body and improve exercise capacity[6].

The heat stress may lead to the occurrence of intestinal disorder and damage to intestinal mucosal barrier,resulting in the digestive disorder,alabsorption and reduced immunity of intestinal mucosa,finally reducing the performance and immunity of livestock.IELs partly reflect the status of intestinal mucosal immunity.This study investigated the pharmacal effects of Chinese herbal medicines through observing the variation in numbers of intestinal epithelial lymphocytes and goblet cells and the effects of herbal medicines on numbers of intestinal epithelial lymphocytes and goblets cells of cocks under heat stress.

Material and Methods

Material and methods

Experimental animalA total of 180 88-d-old healthy Esa Brown cocks,provided by the pasture of Hebei Normal University of Science and Technology,were selected.The cocks were randomly divided into 9 groups with 20 cocks per group.

Main instruments and equipments

The main instruments and equipments used in this study included electronic scales,electric incubator,paraffin slicing machine,sharpener,electric-heated thermostatic water bath and digital microscope.

Main reagentsThe main reagents used in this study included Bouin’s fixative,graded alcohols,xylene,hematoxylin-eosin stain,protein glycerol,acetic acid,paraffin wax,gum,etc.

Experimental design

Experimental animal and feeding conditionsThe 88-d-old Esa Brown cocks were bred according to the conventional feeding and management measures in cages.The cages were placed in the artificial environment laboratory with 16 h of artificial and natural lighting per day.In the laboratory,the changes in temperature in the high temperature season were simulated with stove and electrical oil heater.According to the breeding standards for layers,the daily feed intake was controlled at 75 g/cock,which was completed 3 times a day.At the same time,sufficient clean drinking water was provided to the layers.The manure was cleaned once a day to ensure air quality inside the laboratory.

Grouping of experimental animalThe 180 88-d-old healthy Esa Brown chicken were randomly divided into 9 groups,including High formula I,Moderate formula I,Low formula I,High formula II,Moderate formula II,Low formula II,High temperature control,Room temperature control and VC addition group.The formula I and formula II were to add Chinese herbal extracts to the diet of cocks with 3 gradient doses(high,moderate and low).The addition amount of VC was 0.02%of feed intake.The oral administration was lasted continuously for 10 d (Table1).At the same time,sufficient water was provided to the cocks in all of the groups.In the Room temperature control group,the temperature was controlled at 22 ℃.In the High temperature control group,the temperature was controlled within 26-33 ℃and the humidity was controlled at 65%(Fig.1).

Sampling and slice preparation

SamplingIn each group,5 cocks were selected randomly and then slaughtered on each of 1st,4th,8thand 10thd since the beginning of experiment.For each cock,2 cm middle duodenum,1 cm jejunum segment near the yolk stalk and 1 cm ileum segment near the ileocecal orifice were sampled.

Table1 Administration doses in experimental groups

Slice preparationThe sampled intestine segments were fixed in Bouin’s fixative for 24 h.Subsequently,they were dehydrated with gradient alcohols and transparentized with xylene.Then they were embedded in paraffin and cut into 5 μm slices.The slices were put on glass slides and stained with HE[7].

Counting methods of lymphocytes and goblet cellsThe prepared glass slides carrying samples were observed under a microscope (10×100).The numbers of villus epithelial lymphocytes and goblet cells in a fixed area (0.8 mm×0.4 mm) were counted.A total of 5 horizons were selected for each sample.Then the averages for each sample and for each group were calculated.The obtained data was further analyzed by using SPSS,and the experimental results were expressed as average value ± standard deviation(x±s).

Results and Analysis

Lymphocytes

DuodenumAs shown in Table2,the number of villus epithelial lymphocytes was reduced continuously under high temperature.The number of epithelial lymphocytes in the High formula II was higher than that in the Room temperature control.On the 10thd,the difference was significant (P ＜0.01)(Fig.2,Fig.3).The number of villus epithelial lymphocytes in the High formula II was also higher than that in the High temperature control on the 10th d(P ＜0.05).The High formula II had more villus epithelial lymphocytes compared to the High formula I and Moderate formula I on the 1std (P ＜0.01).On the 10thd,the number of villus epithelial lymphocytes in the High formula II was significantly higher than those in the Low formula I (P＜0.01),Moderate formula II(P＜0.05) and Low formula II (P＜0.05).The lymphocyte number in the High formula II was significantly higher than that in the VC addition group on the 1std (P＜0.05).The lymphocyte number in the High formula I was higher simultaneously than those in the Moderate formula I and Low formula I on the 8thand 10thd(P＜0.05).There were no significant differences in villus epithelial lymphocytes between High formula I and Moderate formula II,Low formula II and VC addition group(P＞0.05).In addition,there were no significant differences in villus epithelial lymphocytes between Moderate formula I and Low formula I,Moderate formula II,Low formula II,VC addition group,Room temperature control and High temperature control (P ＞0.05).The lymphocyte number in the Low formula I was significantly lower than that in the Moderate formula II on the 10thd (P＜0.05).But there were no significant differences between Low formula I and Low formula II,VC addition group,Room temperature control and High temperature control (P＞0.05).None significant dif-ferences in lymphocyte number were found between Room temperature control and VC addition group and High temperature control or between VC addition group and High temperature control(P＞0.05).

Table2 Changes in numbers of villus epithelial lymphocytes in various segments of small intestine of cocks under heat stress

JejunumIn the Room temperature control,the number of villus epithelial lymphocytes was remained stable with the proceeding of experiment.The lymphocyte number in jejunal villus epithelium in the High formula I was higher than that in the Room temperature control.On the 8thd since the beginning of the experiment,the difference became significant(P＜0.05).The lymphocyte number in the High formula II was higher compared to that in the High temperature control.On the 10thd,the difference became significant(P＜0.01).There were significant differences in villus epithelial lymphocyte number between High formula I and High formula II on the 10thd (P＜0.05),between High formula I and High temperature control on the 8thd (P＜0.05),between High formula I and VC addition group on the 1std (P＜0.05),between High formula II and High temperature control on the 10thd(P＜0.01),between High formula II and VC addition group on the 10thd (P＜0.05) and between High formula I and Low formula I on the 8thd(P＜0.01).

IleumThe number of ileal villus epithelial lymphocytes in the High temperature control tended to be decreased gradually with the proceeding of experiment.The lymphocyte numbers in the High formula I and High formula II were all higher than that in the High temperature control on the 8thd(P＜0.05).There were no significant differences in lymphocyte number between High formula I and VC addition group or between VC addition group and High formula II (P＞0.05).On the 8th d,significant differences in villus epithelial lymphocyte number were found between High formula I and Moderate formula I (P＜0.05),between High formula II and High formula I (P＜0.05),between High formula II and Moderate formula II(P＜0.05) and between High formula I and Low formula I(P＜0.01).

Goblet cells

DuodenumAs shown in Table3,the number of goblet cells in duodenal villus epithelium in the Room temperature control was about 14.Moreover,it was remained stable with the proceeding of experiment.In overall,the numbers of villus epithelial goblet cells in the herbal extract-administered groups were higher than that in the Room temperature control.The goblet cell number in the High temperature control was increased first and then decreased with the proceeding of experiment.It was higher than that in the Room temperature control.On the 8thd,the difference reached the significant level.The number of duodenal villus epithelial goblet cells in the High formula I was high compared to that in the High temperature control.The goblet cell number in the High formula II also tended to be decreased with the proceeding of experiment.But it was higher than that in the Temperature control,and the difference reached the largest (4.3) on the 10thd.The goblet cell number in the High formula II was higher than that in the High formula I by 3.31 on the 1std (P＜0.01).There were no significant differences in duodenal villus epithelial goblet cell number between High formula I and VC addition group.The High formula II had more goblet cells compared to that in the VC addition group on the 8thd (P＜0.05),and the Moderate formula II had more goblet cells compared to that in the Moderate formula I on the 4thd(P＜0.05).However,there were nosignificant differences in goblet cell number between High formula I and Moderate formula I (P＞0.05) or between High formula II and Moderate formula II(P＞0.05)on the 10thd.

Table3 Changes in numbers of villus epithelial goblet cells in various segments of small intestine of cocks under heat stress

JejunumThe number of goblet cells in jejunal villus epithelium in the Room temperature control was about 16,and it was remained stable with the proceeding of experiment.In overall,the goblet cell numbers in jejunal villus epithelium in the herbal extract-administered groups were higher than that in the Room temperature control (P ＜0.05) (Fig.4,Fig.5).The goblet cells in the High temperature control was increased first and then decreased,but it was relatively high compared to that in the Room temperature control.There were significant differences in goblet cell number in jejunal villus epithelium between High formula I and High formula II on the 1std (P＜0.05),between High formula I and VC addition group on the 10thd (P ＜0.05) and between High formula II and VC addition group on the 4thd (P＜0.01).However,there were no significant differences in goblet cell number in jejunal villus epithelium between High formula I and Moderate formula I on the 8thd (P＞0.05),between High formula II and Moderate formula II on the 10thd (P ＞0.05) or between Moderate formula I and Moderate formula II on the 10thd(P＞0.05).

IleumThe number of goblet cells in the ileal villus epithelium in the Room temperature control was about 30.The goblet cell numbers in the herbal extract-administered groups were high compared to that in the Room temperature control.However,there were no significant differences in goblet cell number between formula II-added groups and the Room temperature control.The goblet cell number in the High formula I was significantly higher than that in the Room temperature control on the 10thd (P＜0.05) (Fig.6,Fig.7).The goblet cell number in the High temperature control tended to be decreased with the proceeding of experiment,but it was still higher than that in the Room temperature control.The difference was largest(5.2)on the 1std.There were significant differences in ileal villus epithelial goblet cell number between High formula I and High formula II on the 10thd (P＜0.05),between High formula I and Moderate formula I on the 10thd (P＜0.05),between High formula II and VC addition group on the 8thd (P＜0.01),between High formulate II and Moderate formula II on the 10thd (P＜0.01) and between Moderate formula I and Moderate formula II on the 4thd (P ＜0.05).However,there was no significant difference in goblet cell number between High formula I and VC addition group(P＞0.05).Compared to other intestinal segments,ileum had the most goblet cells in villus.

Conclusions and Discussion

Changes in lymphocytes

This study found that under physiological conditions,the number of IELs in chicken accounted for 14%-22% of the number of total intestinal epithelial cells.In addition,the IELs number was decreased gradually from the front end to the terminal end.Under high temperature,the lymphocyte number in mucosal epithelia of chicken tended to be decreased gradually.Compared to Room temperature control,the herbal extract-administered groups could increase the IELs number.In duodenum,the formula I and formula II could significantly increase the IELs number on the 8thand 10thd since the beginning of the experiment.In other intestinal segments,there were significant differences in IELs number between the herbal extractadministered groups and the Room temperature control.Moreover,the increment of formula I was larger than that of formula II.In the High temperature control,the IELs numbers in duodenum,jejunum and ileum were decreased gradually with the proceeding of experiment.In the late experimental period,the formula I and formula all could significantly increase the IELs number.

In intestinal mucosal barrier,the epithelium is mainly composed by epithelial cells,in addition to a large number of dispersed lymphocytes,which are referred to as intestinal epithelial lymphocytes (IELs).The number of IELs is equivalent to that of spleen cells or 40%-50% of the number of lymphocytes in the peripheral circulation.The IELs have a strong immune function.The IELs consist mostly of CD8+T lymphocytes.But the quantity of CD4+T lymphocytes is very small.There are obvious differences between intestinal intraepithelial lymphocytes and peripheral T lymphocytes.Therefore,the changes in number of IELs can reflect the status of intestinal mucosal immunity from certain aspect.IELs,as a special lymphocyte population,are long-termly contacted with normal intestinal flora and pathogenic microorganisms.They play an important role in mucosal immunity against infection,maintainment of the integrity of epithelial cells and regulation of immune responses caused by foreign antigens.

Chinese herbal medicine can improve the immunity and enhance the adaptability of bodies to high temperature environments.It can also enhance the body’s non-specific immune function by increasing the number of cells in the peripheral blood and enhancing the phagocytosis of reticuloendothelial system.

Studies have shown that under high temperature,Chinese herbal medicine can protect IELs from damage through fighting the invasion of free radicals and some inflammatory cytokines,resulting in the increase of IELs number,thereby improving the body’s immunity of animals.

Changes in goblet cells

In this study,the goblet cell numbers in duodenal villus epithelium in the herbal extract-administered groups were significantly higher than that in the control.It was indicated the herbal extract can promote the increase of goblet cell number in duodenal villus epithelium,thereby enhancing the body’s immunity.The goblet cell numbers in herbal extract-administered groups were higher than that in the control.The overall difference reached the significant level.The goblet cell number in the High temperature control was higher than that in the Room temperature control.In duodenum,the High temperature control showed the most goblet cells,which was higher than that in the Room temperature control by 3.6 on the 8thd since the beginning of the experiment (P＜0.01).In Jejunum,there were no significant differences in goblet cell number between High temperature control and Room temperature control.In ileum,the High temperature control showed the most goblet cells on the 1std,which was more than that in the Room temperature control by 5.2 (P＞0.05).The formula I and formula II all could promote the generation of goblet cells,so that the intestinal goblet cells were maintained at a high steady state.The effect of formula II was better compared to those of other groups.With the proceeding of experiment and the prolonging of heat stressed time,the body’s resistance was weakened,so the intestinal villus epithelial goblet cells showed a decreasing trend.

Heat stress has multifaceted effects on animal intestinal function.Under conditions of high temperature,the sympathetic inside bodies of animals will be excited,the secretion of norepinephrine will be increased,but the secretion of thyroxine and motilin will be reduced.The changes above all can slow the intestinal peristalsis.Lin et al.found that the emptying of chime in small intestine of broilers was significantly reduced under 12 h heat stress at 32 ℃[9].The delayed gastrointestinal emptying may result in digesta retention.Thus the digesta cannot be effectively digested or absorbed.On the other hand,digesta retention may cause intestinal flora disorder.The changes in intestinal flora may further increase the invasion of intestinal bacteria,proinflammatory cytokines,such as endotoxin and products of intestinal bacteria and the permeability of intestinal mucosa.In addition,due to the requirements by evaporative heat dissipation,the body’s blood inside chicken will be redistributed,resulting in the increased blood flow in skin and other parts and the relatively decreased blood flow in internal organs and intestine.It has been confirmed that in a burned animal model,the reduced administration flow and reduced blood flow could promote the generation of a large number of oxygen free radicals,resulting in the damage to intestinal capillaries and villus.Under this condition,a large amount of intestinal toxins might invade into blood circulation,aggravating the damage to intestinal tract[10].

The results of this study showed that high environmental temperature promoted the proliferation of intestinal goblet cells in chicken; with the prolonging of heat stressed time,the number of intestinal goblet cells tended to be decreased; formula I and formula II all could promote the generation of intestinal goblet cells,thereby maintaining the intestinal goblet cells at a high steady state;the effect of formula II was better than that of formula I.In conclusion,under high temperature,Chinese herbal medicine additives can promote the generation of goblet cells in intestinal mucosa of chicken,resulting in the secretion of large amounts of mucin,thereby protecting the intestinal mucosa.

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