Mechanism Of Supplementary Sijunzi Decoction On Immune Function In Spleen Deficiency Canines

In this experiment,the animal model of spleen deficiency was replicated with senna and treated with supplementary SIJUNZI Decoction(SJZD).The purpose was to reveal the pathogenesis of spleen deficiency syndrome and the mechanism of supplementary SJZD,and to provide reference for the treatment of spleen deficiency syndrome by supplementary SJZD.24 one-year-old healthy beagle canines were randomly divided into control group,spleen deficiency group,natural recovery group and supplementary SJZD group,six canines in each group.The senna solution was used to replicate spleen deficiency model canines.The spleen deficiency model was established on 7th day,then the canines in spleen deficiency group were killed for collecting samples.The canines in the control group and the natural recovery group were fed a basal diet,the canines in supplementary SJZD group were fed a basal diet with 2%supplementary SJZD.The trial lasted for 14 day.The blood samples were taken at the 0,7th and 14th day,and the levels of serum IFN-?,IL-2,IL-4,IL-6 were detected by enzyme linked immunosorbent assay(ELISA).On the 14th day,all the canines were killed for collecting samples,and the spleen index was calculated,the spleen Tuftsin level was detected,the microscopic and ultrastructural changes in spleen were observed.The duodenum,jejunum and ileum were collected.The protein positive expression level of sIgA,tight junction protein ZO-1 and Occludin were measured in different segments of small intestine by Immunohistochemistry(IHC),the relative expression values of IgA,IgG,IgM and tight junction proteins ZO-1 and Occludin mRNA were measured by fluorescent quantitative PCR,and the expression levels of related proteins Ras,MEK1,ERK1/2 and p-ERK1/2 in the ERK/MAPK pathway were measured by Western blot(WB).The results obtained were as follows:1.At 7th day,compared with the canines of control group,the levels of serum IL-4 and IL-6 in the canines of spleen deficiency group,the natural recovery group and the supplementary SJZD group were significantly increased(P<0.05),and the levels of IFN-y and IL-2 were significantly decreased(P<0.05).At 14th day,compared with the canines in control group,the levels of serum IL-4 and IL-6 in the canines of natural recovery group decreased slightly(P<0.05),and the levels of IFN-? and IL-2 increased slightly(P<0.05).The levels of serum IL-4 and IL-6 in the canines of supplementary SJZD group decreased significantly,and the levels of IFN-y and IL-2 were significantly increased,the indexes of the canines were not significantly different from the control group(P>0.05).2.Compared with canines of control group,the spleen index and spleen Tuftsin level in the canines of spleen deficiency group were extremely significantly decreased(P<0.01),in the canines of natural recovery group were significantly decreased(P<0.05),and in the canines of supplementary SJZD group were no significant difference in spleen Tuftsin levels(P>0.05).3.Under light microscope,the spleen tissue structure of the canines in control group were intact,the boundary of red pulp and white pulp was clearly distinguishable.The spleen sinus congestion,lymphocyte reduction in white pulp,and splenic nodule atrophy were observed in the canines of spleen deficiency group.There was no significant difference in the spleen tissue structure between the canines of supplementary SJZD group.Under electron microscope,the structure of spleen lymphocytes in the control group was basically normal,the cells were tightly connected,and the organelles were relatively intact.The spleen lymphocyte nuclear membrane gap was widened,nuclear pyknosis and mitochondrial vacuole-like lesions were observed in the canines of spleen deficiency group.There was no significant difference in the spleen lymphocytes of the canines in supplementary SJZD group compared with the control group,but some cells still showed repairing tracks.4.Compared with the canines of control group,the protein positive expression levels of sIgA,ZO-1 and Occludin in the duodenum of the canines of spleen deficiency group and the natural recovery group were significantly decreased(P<0.01),and the natural recovery group was higher than the spleen deficiency group.The above indexes of the canines in supplementary SJZD group were not significantly different from the control group(P>0.05).The trend of the above indexes in the jejunum and ileum of the experiment canines were consistent with that of the duodenum.5.Compared with the canines of control group,the relative expression levels of IgA,IgG,IgM,ZO-1 and Occludin mRNA in the duodenum of the canines of spleen deficiency group and the natural recovery group were significantly or extremely significantly decreased(P<0.05,P<0.01),and the above indexes of the natural recovery group was higher than that in the spleen deficiency group.The above indexes of the canines in supplementary SJZD group were not significantly different from the control group(P>0.05).The trend of the above indexes in the jejunum and ileum of the experiment canines were consistent with that of the duodenum.6.Compared with the canines of control group,the expression levels of Ras,MEK1,ERK1/2 and p-ERK1/2 in the duodenum of the canines of spleen deficiency group and the natural recovery group were significantly decreased(P<0.01),and the above indexes of the natural recovery group was higher than that in the spleen deficiency group.The above indexes of the canines in supplementary SJZD group were not significantly different from the control group(P>0.05).The trend of the above indexes in the jejunum and ileum of the experiment canines were consistent with that of the duodenum.The experiment confirmed that the serum cytokine levels of the spleen deficiency canines were disordered,the spleen tissue structure was impaired,and the low expression of intestinal immunoglobulin and tight junction protein may be associated with inhibition of the ERK/MAPK pathway.The supplementary SJZD can modulate cytokine levels,repair damaged organs,activate ERK/MAPK pathway,improve intestinal mucosal immunity,and effectively enhance the immune function of spleen deficiency canines.