Research On The Tonifying Spleen Effect Of Codonopsis Pilosula Polysaccharides Based On Mice With Bitter Cold Type Spleen Deficiency

Objective:Aiming to establish spleen-deficiency animal model and explore the changes of spleen deficiency.On the basis of animal model,we focused on the tonifying spleen effect and its mechanism of Codonopsis pilosula polysaccharides on Sennae Folium-induced spleen-deficiency mice.This study will provide a basis for the basic research of the pharmacodynamic substances of Codonopsis pilosula and clinical medication.Methods:Chapter One.Exploration and establishment of spleen deficiency mouse model of purgation with drugs of bitter flavour and cold nature.To screen the model drug,Sennae Folium group and rhubarb group were intragastrically administered 0.4 g/d of Sennae Folium and 0.4 g/d of rhubarb,respectively.To explore the model method,the single factor model was intragastric with0.4 g/d Sennae Folium,and the compound factor model group was gavaged with 0.4 g/d Sennae Folium and exhaustive swimming for 5 weeks.To explore the dose of Sennae Folium,spleen deficiency syndrome was established by high(0.8 g/d)and low(0.4 g/d)doses of Sennae Folium.To explore the period,the samples were collected at the end of 4,5,6 weeks of modeling.The macroscopic signs such as fur,diarrhea,activity and body weight were observed.Serum D-xylose and gastrin(GAS)were determined to evaluate the animal model.Chapter Two.Changes of spleen deficiency model mice induced by drugs of bitter flavour and cold nature.A total of 20 ICR mice were randomly divided into control and model groups according to random number table.Model group were taken 0.4 g/d Sennae Folium,while control group was fed by gavage with equal volume of normal saline.After 5weeks,the general signs of mice were observed.The serum levels of D-xylose,GAS,MTL,SS,VIP,AMS,Ig G,Ig M,AL,TP and Hb were applied for the evaluation of biological characteristics.Morphology changes of the colon were observed by HE staining.Chapter Three.Research on the tonifying spleen effect of Codonopsis pilosula polysaccharides on spleen-deficiency mice induced by Sennae Folium.ICR mice were randomized into control group,model group,CPP groups with high,medium and low doses,and SJZT group.The Sennae Folium-induced mice were used to evaluate the tonifying spleen effect of CPP.After the treatment of CPP for 6 weeks,the general signs of mice were observed.The contents of RBC,GAS,MTL,SS,VIP,AMS,Ig G,Ig M and LPS were determined.Real time PCR were performed to analyze the m RNA expression levels of claudin and occludin in colon tissue.Western blotting was used to detect the protein expression levels of claudin and occludin in colon tissues of mice.Results:Chapter One.Exploration and establishment of spleen deficiency mouse model of purgation with bitter cold.In the selection of bitter and cold nature drugs,after modeling,macroscopic signs of spleen deficiency was found in both rhubarb group and senna leaf group,and serum D-xylose was significantly lower than control group(P<0.05).Body weight of the senna group was significantly lower than that of the control group(P<0.05),and body weight of the rhubarb group was not significantly different from control group.The modeling of Sennae Folium group was more consistent with the clinical symptoms of spleen deficiency.In the selection of modeling methods,both single factor modeling and compound factor modeling showed signs of spleen deficiency.After 5 weeks of modeling,the body weight of the single factor modeling group was significantly lower than control group(P<0.05),but there was no significant difference between the compound factor modeling group and the control group.There was no significant difference in serum D-xylose between the compound factor model group and the control group,but there was significant difference between the single factor model group and the control group.Compared with single factor modeling,the compound factor modeling method is complicated in operation and the symptoms of spleen deficiency appear more slowly,so the single factor modeling method is chosen.During the experiment of gavage dose,spleen deficiency appeared in both the high and low dose groups,while death,gastric bleeding and intestinal tissue adhesion appeared in the high dose group at the later stage of modeling.The low dose group was safer and more effective,so the low dose was chosen as the modeling dose.During the experiment of the period of modeling,the serum D-xylose content at 4,5and 6 weeks was decreased gradually in the model group,and there was a significant difference between the model group and the blank group at 5 and 6 weeks(P<0.05).The serum gastrin content was compared at 4,5 and 6 weeks after intragastric administration.At the end of 5 week,compared with the control group,the gastrin level of the model group was significantly elevated(P<0.05),so the intragastric administration period of the spleen deficiency model was determined to be 5 weeks.Based on the above results,we concluded that gavage with a dose of 0.4 g Sennae Folium for each mouse every day for 5 weeks was the acceptable approach to create a mice model of spleen deficiency syndrome.Chapter Two.Biologic indicator changes of spleen deficiency model mice.In the model group,the symptoms of spleen deficiency appeared in the late stage,such as bunching,lazy movement,perianal filth,sparse back hair and lackluster.The serum level of D-xylose,VIP,SS,AMS,Ig G and Ig M in model group were significantly decreased compared with the control group(P<0.05),and the serum level of GAS and MTL were significantly increased compared with the control group(P < 0.05).Meanwhile,the mucosa of colon tissue was obviously damaged.The significant increase of serum TP,AL and Hb concentrations may be caused by diarrhea and dehydration caused by senna leaf gavage,which has no correlation with the function of spleen in traditional Chinese medicine,and is not considered to be the organism change caused by spleen deficiency.Chapter Three.Research on the tonifying spleen effect of Codonopsis pilosula polysaccharides on spleen-deficiency mice induced by bitter cold.The serum level of D-xylose,VIP,SS,AMS,Ig G,Ig M,and RBC in Codonopsis pilosula polysaccharides group were significantly increased compared with the model group(P<0.05),and the serum level of GAS,MTL,LPS were significantly decreased compared with the model group(P < 0.05).Meanwhile,the m RNA and protein expressions of claudin and occludin in Codonopsis pilosula polysaccharides group were significantly elevated compared with the model group.Conclusion:1.Intragastric gavage of Sennae Folium(0.4 g/d)for 5 weeks was a more suitable method to construct the spleen deficiency mouse model.Symptom evaluation,serum D-xylose and serum gastrin are the important indexes to evaluate the replication success of spleen deficiency mouse model induced by bitter flavor and cold nature drug.2.The mice with spleen deficiency induced by bitter cold diarrhea showed macro changes such as huddling together,weight increased slowly and objective index changes such as gastrointestinal dysfunction,colonic mucosa injury,and decreased immune function,which can determine the success of the animal model of spleen deficiency caused by bitter cold diarrhea3.Codonopsis pilosula polysaccharides exhibited the significant tonifying effect on Sennae Folium-induced spleen-deficiency mice,which suggested that Codonopsis pilosula polysaccharides was the potential substances for the traditional function of Codonopsis Radix.And its mechanism is related to the regulation of gastrointestinal hormone secretion,enhancing immunity and protect the intestinal mucosal barrier.