Mechanism Of Supplementary Sijunzi Decoction On Digestive Function In Splenic Asthenia Canines

In this experiment,supplementary Sijunzi decoction were added into the diet of canines in order to investigate the effects on physical signs and body weight,physiological and biochemical indexes,pancreatic digestive enzyme activities,tissue morphology and digestive and absorptive functions of small intestine,the expression levels of epidermal growth factor(EGF)and somatostatin(SS)and thier mRNA relative expression values in small intestine,intestinal flora diversity in splenic asthenia canines.Twenty-four one-year-old beagles were selected and randomly divided into four groups,six canines per group.The four groups were control group,splenic asthenia group,natural recovery group and Chinese medicine group,respectively.The canines in control group were fed a basal diet,and in the other three groups were fed with a basal diet and taken with senna decoction at 6 mL/kg a day,one time in the morning and afternoon,to establish spleen deficiency model.After the model developed,the canines in splenic asthenia group were dissected for collecting samples,the canines in Chinese medicine group were fed a basal diet with 1mL/50g supplementary Sijunzi decoction,and the canines in natural recovery group were fed a basal diet.The physical signs of canines in each group were observed during the experiment.On the 0,7,14th day,blood samples were taken from canines to measure blood routine indexes(white blood cell(WBC)count,lymphocyte(LYM)count,neutrophilic granulocyte(Neut)count,red blood cell(RBC)count,hemoglobin(HGB)content,erythrocyte(HCT)and platelets(PLT)count),and sera were separated to measure biochemical indexes(alanine aminotransferase(ALT)activities,aspartate aminotransferase(AST)activities,total protein(TP)content,albumin(ALB)content,blood glucose(GLU)content,total bilirubin(TBIL)content)and the D-xylose content.On the 14th day,all canines were dissected for collecting pancreas tissues in liquid nitrogen to detect the pancreatic digestive enzyme activities.The duodenum,jejunum and ileum were collected and divided into several parts,then kept in paraformaldehyde,glutaraldehyde and liquid nitrogen.The tissues in stationary liquid were used to measure villus length,crypt depth and V/C values by HE staining,to detect protein expression of EGF and SS in small intestine by IHC and to observe the small intestine morphology by electron microscope.The tissues in liquid nitrogen were used to measure the mRNA relative expression levels of EGF and SS by qRT-PCR and protein expression of EGF and SS by WB.Rectum contents were collected for intestinal flora diversity analysis by Illumina sequencing.The results showed that:On the 7th day,after the canines were taking with senna decoction,the spleen deficiency syndrome was observed in test canines,indicating that the spleen deficiency model was successfully developed.1.On the 0 day,there was no significant difference in all indicators of canines in each group.On the 7th day,compared with the canines in control group,body weight of splenic asthenia canines extremely significantly decreased(P<0.01);WBC count,LYM count,and serum TP content and the]ipase,amylase and trypsin activities in pancreatic tissues significantly decreased(P<0.05),RBC count,HGB content,HCT and serum ALB,GLU)and D-xylose contents extremely significantly decreased(P<0.01),aspartate aminotransferase(AST)activies significantly increased(P<0.05)and total bilirubin(TBIL)content extremely significantly increased(P<0.01).2.The structural damage and cell necrosis of microvillus in small intestines were occurred in the splenic asthenia group by scanning electron microscope.Compared with the canines in control group,villus length of splenic asthenia canines extremely significantly decreased(P<0.01),crypt depth extremely significantly increased(P<0.01),V/C extremely significantly decreased(P<0.01).3.Compared with the canines in control group,EGF mRNA relative expression levels of splenic asthenia canines in small intestine significantly decreased(P<0.05)and EGF protein expression in small intestine extremely significantly decreased(P<0.01).SS mRNA relative expression levels of splenic asthenia canines in small intestine significantly increased(P<0.05),SS protein expression in duodenum and jejunum extremely significantly increased(P<0.01)and in ileum significantly increased(P<0.05).4.1)By analyzing the Chao index,ACE index,Shannon index and Simpson index of bacteria in feces sample,it was found that the diversity of intestinal flora in the spleen-deficiency group was lower than that in the control group.2)Compared with the canines in control group,the number of Bacteroidetes in splenic asthenia group significantly decreased(P<0.05),the number of Fusobacteria and Proteobacteria significantly increased(P<0.05);the number of Bacteroidia significantly decreased(P<0.05),the number of Fusobacteriia significantly increased(P<0.05);the number of Bacteroidales significantly decreased(P<0.05),the number of Fusobacteriales significantly increased(P<0.05);the number of Fusobacteriaceae significantly increased(P<0.05),the number of Paraprevotellaceae significantly decreased(P<0.05);the number of Fusobacterium significantly increased(P<0.05),the number of Prevotella group significantly decreased(P<0.05).On the 14 day,after treatment by supplementary Sijunzi decoction,the above indexes of the splenic asthenia canines recovered close to the control group.The results showed that Supplementary Sijunzi decoction can improve the physical signs and body weight,physiological and biochemical indexes,tissue morphology and digestive and absorptive functions of small intestine,the expression levels of epidermal growth factor(EGF)and somatostatin(SS)and thier mRNA relative expression values in small intestine,intestinal flora of splenic asthenia group,and has obvious effects on treating the splenic asthenia canines.