Effect Of Lactobacillus Rhamnosus GG On The Number And Differentiation Of T Lymphocytes In The Intestine Of Early Weaned Piglets

Probiotics are defined by the Food and Agriculture Organization(FAO)of the United Nations and the World Health Organization(WHO),as living microorganisms that when administered in sufficient amounts,confer a health beneficial effect on the human and animal body.Probiotics can be used to prevent and treat intestinal infections,genitourinary diseases,bacterial vaginosis and skin diseases.How probiotics improve the body’s immunity is one of the hotspots of current research.At present the most commonly used probiotic strain is Lactobacillus,a model probiotic that can colonize the intestinal wall,that is critical for the prevention and treatment of various intestinal inflammations in humans and animals.With the continuous development and progress of China’s animal husbandry,the weaning age of piglets is gradually advanced.The immunity of newborn piglets is low,and the disorder of intestinal flora is the cause of diarrhea in piglets.T cells are an important indicator of the body’s immune function.Therefore,this experiment explored the effect of LGG on T lymphocytes in piglets.It aims to lay a theoretical foundation for the development of animal microecological preparations based on lactic acid bacteria and open up new ideas for preventing piglet diarrhea.The specific research contents and results are as follows:(1)In this study,LGG ATCC53103 was selected as the experimental strain.Sixty-day healthy piglets were randomly divided into two groups,the experimental group(LGG group)and the control group(saline group).The LGG group piglets were given 1 mL LGG(6×10~9 CFU/mL)per day.The saline group was given 1 mL of normal saline per day.Piglets were slaughtered on the 15th day,and the mesenteric lymph nodes(MLN),ileum and Peyer’s patches(PPs),ileum and jejunal lamina propria(LP)were taken.The expression of CD3~+CD4~+T lymphocytes and CD4~+CD8~+double positive T lymphocytes were detected by flow cytometry and immunofluorescence.CD3~+CD4~+T lymphocytes were sorted and RNA was extracted,and the expression of CD3~+CD4~+T lymphocyte transcription factor was detected by real-time PCR.The results showed that the proliferation of MTS lymphocytes showed in LGG group was significantly higher than that in normal saline group(P<0.001).The results of flow cytometry showed that there was no significant difference in CD3~+CD4~+T lymphocytes in the MLN of the LGG group compared with the saline group(P>0.05).The CD3~+CD4~+T lymphocytes in the jejunum and ileum of the LGG group were significantly different from those in the saline group(P<0.05)(P<0.05).The CD3~+CD4~+T lymphocytes in the jejunum LP of piglets in LGG group were significantly different from those in saline group(P<0.05).In the LGG group,CD3~+CD4~+T lymphocytes in the ileal LP of piglets were significantly different from those in the saline group(P<0.01).The CD4~+CD8~+T lymphocytes in the IEL of the LGG group were significantly higher than those in the saline group(P<0.001).The results of immunofluorescence showed that the CD3~+CD4~+T lymphocytes in the ileum of the LGG group were more than those in the saline group,and the CD3~+CD4~+T lymphocytes in the PPs were more than those in the saline group.In addition,we selected CD3~+CD4~+T lymphocytes from piglet MLN by flow cytometry,and detected the expression of transcription factors by real-time PCR.The results showed that the transcription factor T-bet of LGG group was significantly higher than GATA(P<0.01).The difference between Foxp3~+and ROR in LGG group was not significant(P>0.05),so T cells tend to differentiate into Th1.In summary,LGG can stimulate the increase in the number of T lymphocytes in piglets and tend to differentiate into Th1.(2)In this study,LGG ATCC53103 was selected as the experimental strain.Twelve healthy piglets were randomly divided into two groups,the experimental group(LGG group)and the control group(saline group).The LGG group piglets were given 1 mL LGG(6×10~9 CFU/mL)per day.The saline group was given 1 mL of normal saline per day.The piglets were slaughtered at 30d and 45d,respectively,and the MLN,jejunum and ileum PPs,LP of the piglets were taken.The expression of CD3~+CD4~+T lymphocytes was detected by flow cytometry,and intestinal villi development was observed by intestinal paraffin section.The results showed that the flow cytometry results at 30d and 45d showed that the CD3~+CD4~+T lymphocytes in the MLN group were significantly different from those in the saline group(P<0.05,P<0.01).The CD3~+CD4~+T lymphocytes in the jejunum PP of piglets in LGG group were significantly different from those in saline group(P<0.05).The CD3~+CD4~+T lymphocytes in the ileum PP of the LGG group were significantly different from those in the saline group(P<0.05,P<0.01).CD3~+CD4~+T lymphocytes and normal saline in the piglets of the LGG group There was a significant difference between the two groups(P<0.001,P<0.05).The length of ileal villi in the LGG group was significantly different from that in the saline group(P<0.01,P<0.001).HE staining results showed The length of the LGG group ileal villi in piglets at 30d,45d was significantly different from that in normal saline group(P<0.01,P<0.01).In LGG group at 15d,30d,45d MLN,jejunum PP,ileum PP,LP There was no significant difference in CD3~+CD4~+T lymphocytes(P>0.05).In summary,LGG can also regulate the proliferation of T lymphocytes in the intestine of early weaned piglets at 30d and 45d,increase the number of CD3~+CD4~+T lymphocytes.The role of intestinal villus development and integrity.(3)In order to analyze the immunological role of the LGG secreted protein P40,we synthesized the P40 protein gene,then the gene linked to the pET28a vector,and purified the P40 protein through a nickel column.A piglet was randomly selected to isolate lymphocytes.The cells were stimulated with LGG,P40,and T cell activation inhibitor(CSA),respectively,and the expression of CD3~+CD4~+T lymphocytes was detected by flow cytometry.The prokaryotic expression system pET28a-P40 was successfully constructed and the P40 protein was successfully purified,and the piglet lymphocytes were isolated.Flow cytometry results showed that CSA was effective in inhibiting T lymphocyte activation.At 12h,24h,36h,the CD3~+CD4~+T lymphocytes in the lymphocytes of LGG group and P40 group were significantly higher than those in the control group(P<0.001).At 12h,24h,36h,LGG+CSA group and P40+CSA group were also significantly higher than CSA group(P<0.05).There was no significant difference between LGG and P40 group(P>0.05).It is thus known that the LGG protein P40 plays a major role in regulating the changes of T lymphocytes in piglets.LGG regulates T lymphocytes in piglets to make T lymphocytes tend to differentiate into Th1.At the same time,LGG regulates the T lymphocytes of piglets to protect the development and integrity of intestinal villi,and the P40 protein secreted by LGG played a key role.